Human taste-specific receptor TIR3

ABSTRACT

The invention provides human G-protein coupled receptors (GCREC) and polynucleotides which identify and encode GCREC. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with aberrant expression of GCREC.

TECHNICAL FIELD

This invention relates to nucleic acid and amino acid sequences ofG-protein coupled receptors and to the use of these sequences in thediagnosis, treatment, and prevention of cell proliferative,neurological, cardiovascular, gastrointestinal, autoimmune/inflammatory,and metabolic disorders, and viral infections, and in the assessment ofthe effects of exogenous compounds on the expression of nucleic acid andamino acid sequences of G-protein coupled receptors.

BACKGROUND OF THE INVENTION

Signal transduction is the general process by which cells respond toextracellular signals. Signal transduction across the plasma membranebegins with the binding of a signal molecule, e.g., a hormone,neurotransmitter, or growth factor, to a cell membrane receptor. Thereceptor, thus activated, triggers an intracellular biochemical cascadethat ends with the activation of an intracellular target molecule, suchas a transcription factor. This process of signal transduction regulatesall types of cell functions including cell proliferation,differentiation, and gene transcription. The G-protein coupled receptors(GPCRs), encoded by one of the largest families of genes yet identified,play a central role in the transduction of extracellular signals acrossthe plasma membrane. GPCRs have a proven history of being successfultherapeutic targets.

GPCRs are integral membrane proteins characterized by the presence ofseven hydrophobic transmembrane domains which together form a bundle ofantiparallel alpha (α) helices. GPCRs range in size from under 400 toover 1000 amino acids (Strosberg, A. D. (1991) Eur. J. Biochem.196:1-10; Coughlin, S. R. (1994) Curr. Opin. Cell Biol. 6:191-197). Theamino-terminus of a GPCR is extracellular, is of variable length, and isoften glycosylated. The carboxy-terminus is cytoplasmic and generallyphosphorylated. Extracellular loops alternate with intracellular loopsand link the transmembrane domains. Cysteine disulfide bridges linkingthe second and third extracellular loops may interact with agonists andantagonists. The most conserved domains of GPCRs are the transmembranedomains and the first two cytoplasmic loops. The transmembrane domainsaccount, in part, for structural and functional features of thereceptor. In most cases, the bundle of a helices forms a ligand-bindingpocket. The extracellular N-terminal segment, or one or more of thethree extracellular loops, may also participate in ligand binding.Ligand binding activates the receptor by inducing a conformationalchange in intracellular portions of the receptor. In turn, the large,third intracellular loop of the activated receptor interacts with aheterotrimeric guanine nucleotide binding (G) protein complex whichmediates further intracellular signaling activities, including theactivation of second messengers such as cyclic AMP (cAMP), phospholipaseC, and inositol triphosphate, and the interaction of the activated GPCRwith ion channel proteins. (See, e.g., Watson, S. and S. Arkinstall(1994) The G-protein Linked Receptor Facts Book, Academic Press, SanDiego Calif., pp. 2-6; Bolander, F. F. (1994) Molecular Endocrinology,Academic Press, San Diego Calif., pp. 162-176; Baldwin, J. M. (1994)Curr. Opin. Cell Biol. 6:180-190.)

GPCRs include receptors for sensory signal mediators (e.g., light andolfactory stimulatory molecules); adenosine, γ-aminobutyric acid (GABA),hepatocyte growth factor, melanocortins, neuropeptide Y, opioidpeptides, opsins, somatostatin, tachykinins, vasoactive intestinalpolypeptide family, and vasopressin; biogenic amines (e.g., dopamine,epinephrine and norepinephrine, histamine, glutamate (metabotropiceffect), acetylcholine (muscarinic effect), and serotonin); chemokines;lipid mediators of inflammation (e.g., prostaglandins and prostanoids,platelet activating factor, and leukotrienes); and peptide hormones(e.g., bombesin, bradykinin, calcitonin, C5a anaphylatoxin, endothelin,follicle-stimulating hormone (FSH), gonadotropic-releasing hormone(GnRH), neurokinin, and thyrotropin-releasing hormone (TRH), andoxytocin). GPCRs which act as receptors for stimuli that have yet to beidentified are known as orphan receptors.

The diversity of the GPCR family is further increased by alternativesplicing. Many GPCR genes contain introns, and there are currently over30 such receptors for which splice variants have been identified. Thelargest number of variations are at the protein C-terminus. N-terminaland cytoplasmic loop variants are also frequent, while variants in theextracellular loops or transmembrane domains are less common. Somereceptors have more than one site at which variance can occur. Thesplicing variants appear to be functionally distinct, based uponobserved differences in distribution, signaling, coupling, regulation,and ligand binding profiles (Kilpatrick, G. J. et al. (1999) TrendsPharmacol. Sci. 20:294-301).

GPCRs can be divided into three major subfamilies: the rhodopsin-like,secretin-like, and metabotropic glutamate receptor subfamilies. Membersof these GPCR subfamilies share similar functions and the characteristicseven transmembrane structure, but have divergent amino acid sequences.The largest family consists of the rhodopsin-like GPCRs, which transmitdiverse extracellular signals including hormones, neurotransmitters, andlight Rhodopsin is a photosensitive GPCR found in animal retinas. Invertebrates, rhodopsin molecules are embedded in membranous stacks foundin photoreceptor (rod) cells. Each rhodopsin molecule responds to aphoton of light by triggering a decrease in cGMP levels which leads tothe closure of plasma membrane sodium channels. In this manner, a visualsignal is converted to a neural impulse. Other rhodopsin-like GPCRs aredirectly involved in responding to neurotransmitters. These GPCRsinclude the receptors for adrenaline (adrenergic receptors),acetylcholine (muscarinic receptors), adenosine, galanin, and glutamate(N-methyl-D-aspartate/NMDA receptors). (Reviewed in Watson, S. and S.Arkinstall (1994) The G-Protein Linked Receptor Facts Book, AcademicPress, San Diego Calif., pp. 7-9, 19-22, 32-35, 130-131, 214-216,221-222; Habert-Ortoli, E. et al. (1994) Proc. Natl. Acad. Sci. USA91:9780-9783.)

The galanin receptors mediate the activity of the neuroendocrine peptidegalanin, which inhibits secretion of insulin, acetylcholine, serotoninand noradrenaline, and stimulates prolactin and growth hormone release.Galanin receptors are involved in feeding disorders, pain, depression,and Alzheimer's disease (Kask, K. et al. (1997) Life Sci. 60:1523-1533).Other nervous system rhodopsin-like GPCRs include a growing family ofreceptors for lysophosphatidic acid and other lysophospholipids, whichappear to have roles in development and neuropathology (Chun, J. et al.(1999) Cell Biochem. Biophys. 30:213-242).

The largest subfamily of GPCRs, the olfactory receptors, are alsomembers of the rhodopsin-like GPCR family. These receptors function bytransducing odorant signals. Numerous distinct olfactory receptors arerequired to distinguish different odors. Each olfactory sensory neuronexpresses only one type of olfactory receptor, and distinct spatialzones of neurons expressing distinct receptors are found in nasalpassages. For example, the RA1c receptor which was isolated from a ratbrain library, has been shown to be limited in expression to verydistinct regions of the brain and a defined zone of the olfactoryepithelium (Raming, K. et al. (1998) Receptors Channels 6:141-151).However, the expression of olfactory-like receptors is not confined toolfactory tissues. For example, three rat genes encoding olfactory-likereceptors having typical GPCR characteristics showed expression patternsnot only in taste and olfactory tissue, but also in male reproductivetissue (Thomas, M. B. et al. (1996) Gene 178:1-5).

Members of the secretin-like GPCR subfamily have as their ligandspeptide hormones such as secretin, calcitonin, glucagon, growthhormone-releasing hormone, parathyroid hormone, and vasoactiveintestinal peptide. For example, the secretin receptor responds tosecretin, a peptide hormone that stimulates the secretion of enzymes andions in the pancreas and small intestine (Watson, supra, pp. 278-283).Secretin receptors are about 450 amino acids in length and are found inthe plasma membrane of gastrointestinal cells. Binding of secretin toits receptor stimulates the production of cAMP.

Examples of secretin-like GPCRs implicated in inflammation and theimmune response include the EGF module-containing, mucin-like hormonereceptor (Emr1) and CD97 receptor proteins. These GPCRs are members ofthe recently characterized EGF-TM7 receptors subfamily. These seventransmembrane hormone receptors exist as heterodimers in vivo andcontain between three and seven potential calcium-binding EGF-likemotifs. CD97 is predominantly expressed in leukocytes and is markedlyupregulated on activated B and T cells (McKnight, A. J. and S. Gordon(1998) J. Leukoc. Biol. 63:271-280).

The third GPCR subfamily is the metabotropic glutamate receptor family.Glutamate is the major excitatory neurotransmitter in the centralnervous system. The metabotropic glutamate receptors modulate theactivity of intracellular effectors, and are involved in long-termpotentiation (Watson, supra, p. 130). The Ca²⁺-sensing receptor, whichsenses changes hi the extracellular concentration of calcium ions, has alarge extracellular domain including clusters of acidic amino acidswhich may be involved in calcium binding. The metabotropic glutamatereceptor family also includes pheromone receptors, the GABA_(B)receptors, and the taste receptors.

Other subfamilies of GPCRs include two groups of chemoreceptor genesfound in the nematodes Caenorhabditis elegans and Caenorhabditisbriggsae, which are distantly related to the mammalian olfactoryreceptor genes. The yeast pheromone receptors STE2 and STE3, involved inthe response to mating factors on the cell membrane, have their ownseven-transmembrane signature, as do the cAMP receptors from the slimemold Dictyostelium discoideum, which are thought to regulate theaggregation of individual cells and control the expression of numerousdevelopmentally-regulated genes.

GPCR mutations, which may cause loss of function or constitutiveactivation, have been associated with numerous human diseases (Coughlin,supra). For instance, retinitis pigmentosa may arise from mutations inthe rhodopsin gene. Furthermore, somatic activating mutations in thethyrotropin receptor have been reported to cause hyperfunctioningthyroid adenomas, suggesting that certain GPCRs susceptible toconstitutive activation may behave as protooncogenes (Parma, J. et al.(1993) Nature 365:649-651). GPCR receptors for the following ligandsalso contain mutations associated with human disease: luteinizinghormone (precocious puberty); vasopressin V₂ (X-linked nephrogenicdiabetes); glucagon (diabetes and hypertension); calcium(hyperparathyroidism, hypocalcuria, hypercalcernia); parathyroid hormone(short limbed dwarfism); β₃-adrenoceptor (obesity, non-insulin-dependentdiabetes mellitus); growth hormone releasing hormone (dwarfism); andadrenocorticotropin (glucocorticoid deficiency) (Wilson, S. et al.(1998) Br. J. Pharmocol. 125:1387-1392; Stadel, J. M. et al. (1997)Trends Pharmacol. Sci. 18:430-437). GPCRs are also involved indepression, schizophrenia, sleeplessness, hypertension, anxiety, stress,renal failure, and several cardiovascular disorders (Horn, F. and G.Vriend (1998) J. Mol. Med. 76:464-468).

In addition, within the past 20 years several hundred new drugs havebeen recognized that are directed towards activating or inhibitingGPCRs. The therapeutic targets of these drugs span a wide range ofdiseases and disorders, including cardiovascular, gastrointestinal, andcentral nervous system disorders as well as cancer, osteoporosis andendometriosis (Wilson, supra; Stadel, supra). For example, the dopamineagonist L-dopa is used to treat Parkinson's disease, while a dopamineantagonist is used to treat schizophrenia and the early stages ofHuntington's disease. Agonists and antagonists of adrenoceptors havebeen used for the treatment of asthma, high blood pressure, othercardiovascular disorders, and anxiety; muscarinic agonists are used inthe treatment of glaucoma and tachycardia; serotonin 5HT1D antagonistsare used against migraine; and histamine H1 antagonists are used againstallergic and anaphylactic reactions, hay fever, itching, and motionsickness (Horn, supra).

Recent research suggests potential future therapeutic uses for GPCRs inthe treatment of metabolic disorders including diabetes, obesity, andosteoporosis. For example, mutant V2 vasopressin receptors causingnephrogenic diabetes could be functionally rescued in vitro byco-expression of a C-terminal V2 receptor peptide spanning the regioncontaining the mutations. This result suggests a possible novel strategyfor disease treatment (Schoneberg, T. et al. (1996) EMBO J.15:1283-1291). Mutations in melanocortin-4 receptor (MC4R) areimplicated in human weight regulation and obesity. As with thevasopressin V2 receptor mutants, these MC4R mutants are defective intrafficking to the plasma membrane (Ho, G. and R. G. MacKenzie (1999) J.Biol. Chem. 274:35816-35822), and thus might be treated with a similarstrategy. The type 1 receptor for parathyroid hormone (PTH) is a GPCRthat mediates the PTH-dependent regulation of calcium homeostasis in thebloodstream. Study of PTH/receptor interactions may enable thedevelopment of novel PTH receptor ligands for the treatment ofosteoporosis (Mannstadt, M. et al. (1999) Am. J. Physiol.277:F665-F675).

The chemokine receptor group of GPCRs have potential therapeutic utilityin inflammation and infectious disease. (For review, see Locati, M. andP. M. Murphy (1999) Annu. Rev. Med. 50:425-440.) Chemokines are smallpolypeptides that act as intracellular signals in the regulation ofleukocyte trafficking, hematopoiesis, and angiogenesis. Targeteddisruption of various chemokine receptors in mice indicates that thesereceptors play roles in pathologic inflammation and in autoimmunedisorders such as multiple sclerosis. Chemokine receptors are alsoexploited by infectious agents, including herpesviruses and the humanimmunodeficiency virus (HIV-1) to facilitate infection. A truncatedversion of chemokine receptor CCR5, which acts as a coreceptor forinfection of T-cells by HIV-1, results in resistance to AIDS, suggestingthat CCR5 antagonists could be useful in preventing the development ofAIDS.

The discovery of new G-protein coupled receptors and the polynucleotidesencoding them satisfies a need in the art by providing new compositionswhich are useful in the diagnosis, prevention, and treatment of cellproliferative, neurological, cardiovascular, gastrointestinal,autoimmune/inflammatory, and metabolic disorders, and viral infections,and in the assessment of the effects of exogenous compounds on theexpression of nucleic acid and amino acid sequences of G-protein coupledreceptors.

SUMMARY OF THE INVENTION

The invention features purified polypeptides, G-protein coupledreceptors, referred to collectively as “GCREC” and individually as“GCREC-1,” “GCREC-2,” “GCREC-3,” “GCREC-4,” “GCREC-5,” “GCREC-6,”“GCREC-7,” “GCREC-8,” “GCREC-9,” and “GCREC-10.” In one aspect, theinvention provides an isolated polypeptide selected from the groupconsisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1-10, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:1-10, c) a biologically active fragment of a polypeptidehaving an amino acid sequence selected from the group consisting of SEQID NO:1-10, and d) an immunogenic fragment of a polypeptide having anamino acid sequence selected from the group consisting of SEQ IDNO:1-10. In one alternative, the invention provides an isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO:1-10.

The invention further provides an isolated polynucleotide encoding apolypeptide selected from the group consisting of a) a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:1-10, b) a polypeptide comprising a naturally occurring aminoacid sequence at least 90% identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1-10, c) a biologically activefragment of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-10, and d) an immunogenic fragmentof a polypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-10. In one alternative, the polynucleotideencodes a polypeptide selected from the group consisting of SEQ IDNO:1-10. In another alternative, the polynucleotide is selected from thegroup consisting of SEQ ID NO:11-20.

Additionally, the invention provides a recombinant polynucleotidecomprising a promoter sequence operably linked to a polynucleotideencoding a polypeptide selected from the group consisting of a) apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-10, b) a polypeptide comprising a naturallyoccurring amino acid sequence at least 90% identical to an amino acidsequence selected from the group consisting of SEQ ID NO:1-10, c) abiologically active fragment of a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO:1-10, and d) animmunogenic fragment of a polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO:1-10. In onealternative, the invention provides a cell transformed with therecombinant polynucleotide. In another alternative, the inventionprovides a transgenic organism comprising the recombinantpolynucleotide.

The invention also provides a method for producing a polypeptideselected from the group consisting of a) a polypeptide comprising anamino acid sequence selected from the group consisting of SEQ IDNO:1-10, b) a polypeptide comprising a naturally occurring amino acidsequence at least 90% identical to an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-10, c) a biologically activefragment of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-10, and d) an immunogenic fragmentof a polypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-10. The method comprises a) culturing a cellunder conditions suitable for expression of the polypeptide, whereinsaid cell is transformed with a recombinant polynucleotide comprising apromoter sequence operably linked to a polynucleotide encoding thepolypeptide, and b) recovering the polypeptide so expressed.

Additionally, the invention provides an isolated antibody whichspecifically binds to a polypeptide selected from the group consistingof a) a polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-10, b) a polypeptide comprising anaturally occurring amino acid sequence at least 90% identical to anamino acid sequence selected from the group consisting of SEQ IDNO:1-10, c) a biologically active fragment of a polypeptide having anamino acid sequence selected from the group consisting of SEQ IDNO:1-10, and d) an immunogenic fragment of a polypeptide having an aminoacid sequence selected from the group consisting of SEQ ID NO:1-10.

The invention further provides an isolated polynucleotide selected fromthe group consisting of a) a polynucleotide comprising a polynucleotidesequence selected from the group consisting of SEQ ID NO:11-20, b) apolynucleotide comprising a naturally occurring polynucleotide sequenceat least 90% identical to a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:11-20, c) a polynucleotide complementaryto the polynucleotide of a), d) a polynucleotide complementary to thepolynucleotide of b), and e) an RNA equivalent of a)-d). In onealternative, the polynucleotide comprises at least 60 contiguousnucleotides.

Additionally, the invention provides a method for detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide selected from the group consisting of a) apolynucleotide comprising a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:11-20, b) a polynucleotide comprising anaturally occurring polynucleotide sequence at least 90% identical to apolynucleotide sequence selected from the group consisting of SEQ IDNO:11-20, c) a polynucleotide complementary to the polynucleotide of a),d) a polynucleotide complementary to the polynucleotide of b), and e) anRNA equivalent of a)-d). The method comprises a) hybridizing the samplewith a probe comprising at least 20 contiguous nucleotides comprising asequence complementary to said target polynucleotide in the sample, andwhich probe specifically hybridizes to said target polynucleotide, underconditions whereby a hybridization complex is formed between said probeand said target polynucleotide or fragments thereof, and b) detectingthe presence or absence of said hybridization complex, and optionally,if present, the amount thereof. In one alternative, the probe comprisesat least 60 contiguous nucleotides.

The invention further provides a method for detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide selected from the group consisting of a) apolynucleotide comprising a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:11-20, b) a polynucleotide comprising anaturally occurring polynucleotide sequence at least 90% identical to apolynucleotide sequence selected from the group consisting of SEQ IDNO:11-20, c) a polynucleotide complementary to the polynucleotide of a),d) a polynucleotide complementary to the polynucleotide of b), and e) anRNA equivalent of a)-d). The method comprises a) amplifying said targetpolynucleotide or fragment thereof using polymerase chain reactionamplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.

The invention further provides a composition comprising an effectiveamount of a polypeptide selected from the group consisting of a) apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-10, b) a polypeptide comprising a naturallyoccurring amino acid sequence at least 90% identical to an amino acidsequence selected from the group consisting of SEQ ID NO:1-10, c) abiologically active fragment of a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO:1-10, and d) animmunogenic fragment of a polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO:1-10, and apharmaceutically acceptable excipient. In one embodiment, thecomposition comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-10. The invention additionally provides amethod of treating a disease or condition associated with decreasedexpression of functional GCREC, comprising administering to a patient inneed of such treatment the composition.

The invention also provides a method for screening a compound foreffectiveness as an agonist of a polypeptide selected from the groupconsisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1-10, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:1-10, c) a biologically active fragment of a polypeptidehaving an amino acid sequence selected from the group consisting of SEQID NO:1-10, and d) an immunogenic fragment of a polypeptide having anamino acid sequence selected from the group consisting of SEQ IDNO:1-10. The method comprises a) exposing a sample comprising thepolypeptide to a compound, and b) detecting agonist activity in thesample. In one alternative, the invention provides a compositioncomprising an agonist compound identified by the method and apharmaceutically acceptable excipient. In another alternative, theinvention provides a method of treating a disease or conditionassociated with decreased expression of functional GCREC, comprisingadministering to a patient in need of such treatment the composition.

Additionally, the invention provides a method for screening a compoundfor effectiveness as an antagonist of a polypeptide selected from thegroup consisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1-10, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:1-10, c) a biologically active fragment of a polypeptidehaving an amino acid sequence selected from the group consisting of SEQID NO:1-10, and d) an immunogenic fragment of a polypeptide having anamino acid sequence selected from the group consisting of SEQ IDNO:1-10. The method comprises a) exposing a sample comprising thepolypeptide to a compound, and b) detecting antagonist activity in thesample. In one alternative, the invention provides a compositioncomprising an antagonist compound identified by the method and apharmaceutically acceptable excipient. In another alternative, theinvention provides a method of treating a disease or conditionassociated with overexpression of functional GCREC, comprisingadministering to a patient in need of such treatment the composition.

The invention further provides a method of screening for a compound thatspecifically binds to a polypeptide selected from the group consistingof a) a polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-10, b) a polypeptide comprising anaturally occurring amino acid sequence at least 90% identical to anamino acid sequence selected from the group consisting of SEQ IDNO:1-10, c) a biologically active fragment of a polypeptide having anamino acid sequence selected from the group consisting of SEQ IDNO:1-10, and d) an immunogenic fragment of a polypeptide having an aminoacid sequence selected from the group consisting of SEQ ID NO:1-10. Themethod comprises a) combining the polypeptide with at least one testcompound under suitable conditions, and b) detecting binding of thepolypeptide to the test compound, thereby identifying a compound thatspecifically binds to the polypeptide.

The invention further provides a method of screening for a compound thatmodulates the activity of a polypeptide selected from the groupconsisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1-10, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:1-10, c) a biologically active fragment of a polypeptidehaving an amino acid sequence selected from the group consisting of SEQID NO:1-10, and d) an immunogenic fragment of a polypeptide having anamino acid sequence selected from the group consisting of SEQ IDNO:1-10. The method comprises a) combining the polypeptide with at leastone test compound under conditions permissive for the activity of thepolypeptide, b) assessing the activity of the polypeptide in thepresence of the test compound, and c) comparing the activity of thepolypeptide in the presence of the test compound with the activity ofthe polypeptide in the absence of the test compound, wherein a change inthe activity of the polypeptide in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptide.

The invention further provides a method for screening a compound foreffectiveness in altering expression of a target polynucleotide, whereinsaid target polynucleotide comprises a sequence selected from the groupconsisting of SEQ ID NO:11-20, the method comprising a) exposing asample comprising the target polynucleotide to a compound, and b)detecting altered expression of the target polynucleotide.

The invention further provides a method for assessing toxicity of a testcompound, said method comprising a) treating a biological samplecontaining nucleic acids with the test compound; b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide selected from thegroup consisting of i) a polynucleotide comprising a polynucleotidesequence selected from the group consisting of SEQ ID NO:11-20, ii) apolynucleotide comprising a naturally occurring polynucleotide sequenceat least 90% identical to a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:11-20, iii) a polynucleotide having asequence complementary to i), iv) a polynucleotide complementary to thepolynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridizationoccurs under conditions whereby a specific hybridization complex isformed between said probe and a target polynucleotide in the biologicalsample, said target polynucleotide selected from the group consisting ofi) a polynucleotide comprising a polynucleotide sequence selected fromthe group consisting of SEQ ID NO:11-20, ii) a polynucleotide comprisinga naturally occurring polynucleotide sequence at least 90% identical toa polynucleotide sequence selected from the group consisting of SEQ IDNO:11-20, iii) a polynucleotide complementary to the polynucleotide ofi), iv) a polynucleotide complementary to the polynucleotide of ii), andv) an RNA equivalent of i)-iv). Alternatively, the target polynucleotidecomprises a fragment of a polynucleotide sequence selected from thegroup consisting of i)-v) above; c) quantifying the amount ofhybridization complex; and d) comparing the amount of hybridizationcomplex in the treated biological sample with the amount ofhybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.

BRIEF DESCRIPTION OF THE TABLES

Table 1 summarizes the nomenclature for the full length polynucleotideand polypeptide sequences of the present invention.

Table 2 shows the GenBank identification number and annotation of thenearest GenBank homolog for polypeptides of the invention. Theprobability score for the match between each polypeptide and its GenBankhomolog is also shown.

Table 3 shows structural features of polypeptide sequences of theinvention, including predicted motifs and domains, along with themethods, algorithms, and searchable databases used for analysis of thepolypeptides.

Table 4 lists the cDNA and/or genomic DNA fragments which were used toassemble polynucleotide sequences of the invention, along with selectedfragments of the polynucleotide sequences.

Table 5 shows the representative cDNA library for polynucleotides of theinvention.

Table 6 provides an appendix which describes the tissues and vectorsused for construction of the cDNA libraries shown in Table 5.

Table 7 shows the tools, programs, and algorithms used to analyze thepolynucleotides and polypeptides of the invention, along with applicabledescriptions, references, and threshold parameters.

Table 8 shows tissue-specific expression of polynucleotides of theinvention.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular machines, materials and methods described, as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to “ahost cell” includes a plurality of such host cells, and a reference to“an antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any machines,materials, and methods similar or equivalent to those described hereincan be used to practice or test the present invention, the preferredmachines, materials and methods are now described. All publicationsmentioned herein are cited for the purpose of describing and disclosingthe cell lines, protocols, reagents and vectors which are reported inthe publications and which might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

DEFINITIONS

“GCREC” refers to the amino acid sequences of substantially purifiedGCREC obtained from any species, particularly a mammalian species,including bovine, ovine, porcine, murine, equine, and human, and fromany source, whether natural, synthetic, semi-synthetic, or recombinant.

The term “agonist” refers to a molecule which intensifies or mimics thebiological activity of GCREC. Agonists may include proteins, nucleicacids, carbohydrates, small molecules, or any other compound orcomposition which modulates the activity of GCREC either by directlyinteracting with GCREC or by acting on components of the biologicalpathway in which GCREC participates.

An “allelic variant” is an alternative form of the gene encoding GCREC.Allelic variants may result from at least one mutation in the nucleicacid sequence and may result in altered mRNAs or in polypeptides whosestructure or function may or may not be altered. A gene may have none,one, or many allelic variants of its naturally occurring form. Commonmutational changes which give rise to allelic variants are generallyascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

“Altered” nucleic acid sequences encoding GCREC include those sequenceswith deletions, insertions, or substitutions of different nucleotides,resulting in a polypeptide the same as GCREC or a polypeptide with atleast one functional characteristic of GCREC. Included within thisdefinition are polymorphisms which may or may not be readily detectableusing a particular oligonucleotide probe of the polynucleotide encodingGCREC, and improper or unexpected hybridization to allelic variants,with a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding GCREC. The encoded protein may also be“altered,” and may contain deletions, insertions, or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent GCREC. Deliberate amino acid substitutions maybe made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues, as long as the biological or immunological activity of GCRECis retained. For example, negatively charged amino acids may includeaspartic acid and glutamic acid, and positively charged amino acids mayinclude lysine and arginine. Amino acids with uncharged polar sidechains having similar hydrophilicity values may include: asparagine andglutamine; and serine and threonine. Amino acids with uncharged sidechains having similar hydrophilicity values may include: leucine,isoleucine, and valine; glycine and alanine; and phenylalanine andtyrosine.

The terms “amino acid” and “amino acid sequence” refer to anoligopeptide, peptide, polypeptide, or protein sequence, or a fragmentof any of these, and to naturally occurring or synthetic molecules.Where “amino acid sequence” is recited to refer to a sequence of anaturally occurring protein molecule, “amino acid sequence” and liketerms are not meant to limit the amino acid sequence to the completenative amino acid sequence associated with the recited protein molecule.

“Amplification” relates to the production of additional copies of anucleic acid sequence. Amplification is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art.

The term “antagonist” refers to a molecule which inhibits or attenuatesthe biological activity of GCREC. Antagonists may include proteins suchas antibodies, nucleic acids, carbohydrates, small molecules, or anyother compound or composition which modulates the activity of GCRECeither by directly interacting with GCREC or by acting on components ofthe biological pathway in which GCREC participates.

The term “antibody” refers to intact immunoglobulin molecules as well asto fragments thereof, such as Fab, F(ab′)₂, and Fv fragments, which arecapable of binding an epitopic determinant. Antibodies that bind GCRECpolypeptides can be prepared using intact polypeptides or usingfragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

The term “antigenic determinant” refers to that region of a molecule(i.e., an epitope) that makes contact with a particular antibody. When aprotein or a fragment of a protein is used to immunize a host animal,numerous regions of the protein may induce the production of antibodieswhich bind specifically to antigenic determinants (particular regions orthree-dimensional structures on the protein). An antigenic determinantmay compete with the intact antigen (i.e., the immunogen used to elicitthe immune response) for binding to an antibody.

The term “antisense” refers to any composition capable of base-pairingwith the “sense” (coding) strand of a specific nucleic acid sequence.Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA);oligonucleotides having modified backbone linkages such asphosphorothioates, methylphosphonates, or benzylphosphonates;oligonucleotides having modified sugar groups such as 2′-methoxyethylsugars or 2′-methoxyethoxy sugars; or oligonucleotides having modifiedbases such as 5-methyl cytosine, 2′-deoxyuracil, or7-deaza-2′-deoxyguanosine. Antisense molecules may be produced by anymethod including chemical synthesis or transcription. Once introducedinto a cell, the complementary antisense molecule base-pairs with anaturally occurring nucleic acid sequence produced by the cell to formduplexes which block either transcription or translation. Thedesignation “negative” or “minus” can refer to the antisense strand, andthe designation “positive” or “plus” can refer to the sense strand of areference DNA molecule.

The term “biologically active” refers to a protein having structural,regulatory, or biochemical functions of a naturally occurring molecule.Likewise, “immunologically active” or “immunogenic” refers to thecapability of the natural, recombinant, or synthetic GCREC, or of anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

“Complementary” describes the relationship between two single-strandednucleic acid sequences that anneal by base-pairing. For example,5′-AGT-3′ pairs with its complement, 3′-TCA-5′.

A “composition comprising a given polynucleotide sequence” and a“composition comprising a given amino acid sequence” refer broadly toany composition containing the given polynucleotide or amino acidsequence. The composition may comprise a dry formulation or an aqueoussolution. Compositions comprising polynucleotide sequences encodingGCREC or fragments of GCREC may be employed as hybridization probes. Theprobes may be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., sodium dodecyl sulfate; SDS), and other components(e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).

“Consensus sequence” refers to a nucleic acid sequence which has beensubjected to repeated DNA sequence analysis to resolve uncalled bases,extended using the XL-PCR kit (Applied Biosystems, Foster City Calif.)in the 5′ and/or the 3′ direction, and resequenced, or which has beenassembled from one or more overlapping cDNA, EST, or genomic DNAfragments using a computer program for fragment assembly, such as theGELVIEW fragment assembly system (GCG, Madison Wis.) or Phrap(University of Washington, Seattle Wash.). Some sequences have been bothextended and assembled to produce the consensus sequence.

“Conservative amino acid substitutions” are those substitutions that arepredicted to least interfere with the properties of the originalprotein, i.e., the structure and especially the function of the proteinis conserved and not significantly changed by such substitutions. Thetable below shows amino acids which may be substituted for an originalamino acid in a protein and which are regarded as conservative aminoacid substitutions.

Original Residue Conservative Substitution Ala Gly, Ser Arg His, Lys AsnAsp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln,His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, Val Lys Arg,Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser,Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu, Thr

Conservative amino acid substitutions generally maintain (a) thestructure of the polypeptide backbone in the area of the substitution,for example, as a beta sheet or alpha helical conformation, (b) thecharge or hydrophobicity of the molecule at the site of thesubstitution, and/or (c) the bulk of the side chain.

A “deletion” refers to a change in the amino acid or nucleotide sequencethat results in the absence of one or more amino acid residues ornucleotides.

The term “derivative” refers to a chemically modified polynucleotide orpolypeptide. Chemical modifications of a polynucleotide can include, forexample, replacement of hydrogen by an alkyl, acyl, hydroxyl, or aminogroup. A derivative polynucleotide encodes a polypeptide which retainsat least one biological or immunological function of the naturalmolecule. A derivative polypeptide is one modified by glycosylation,pegylation, or any similar process that retains at least one biologicalor immunological function of the polypeptide from which it was derived.

A “detectable label” refers to a reporter molecule or enzyme that iscapable of generating a measurable signal and is covalently ornoncovalently joined to a polynucleotide or polypeptide.

“Differential expression” refers to increased or upregulated; ordecreased, downregulated, or absent gene or protein expression,determined by comparing at least two different samples. Such comparisonsmay be carried out between, for example, a treated and an untreatedsample, or a diseased and a normal sample.

A “fragment” is a unique portion of GCREC or the polynucleotide encodingGCREC which is identical in sequence to but shorter in length than theparent sequence. A fragment may comprise up to the entire length of thedefined sequence, minus one nucleotide/amino acid residue. For example,a fragment may comprise from 5 to 1000 contiguous nucleotides or aminoacid residues. A fragment used as a probe, primer, antigen, therapeuticmolecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25,30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotidesor amino acid residues in length. Fragments may be preferentiallyselected from certain regions of a molecule. For example, a polypeptidefragment may comprise a certain length of contiguous amino acidsselected from the first 250 or 500 amino acids (or first 25% or 50%) ofa polypeptide as shown in a certain defined sequence. Clearly theselengths are exemplary, and any length that is supported by thespecification, including the Sequence Listing, tables, and figures, maybe encompassed by the present embodiments.

A fragment of SEQ ID NO:11-20 comprises a region of uniquepolynucleotide sequence that specifically identifies SEQ ID NO:11-20,for example, as distinct from any other sequence in the genome fromwhich the fragment was obtained. A fragment of SEQ ID NO:11-20 isuseful, for example, in hybridization and amplification technologies andin analogous methods that distinguish SEQ ID NO:11-20 from relatedpolynucleotide sequences. The precise length of a fragment of SEQ IDNO:11-20 and the region of SEQ ID NO:11-20 to which the fragmentcorresponds are routinely determinable by one of ordinary skill in theart based on the intended purpose for the fragment.

A fragment of SEQ ID NO:1-10 is encoded by a fragment of SEQ IDNO:11-20. A fragment of SEQ ID NO:1-10 comprises a region of uniqueamino acid sequence that specifically identifies SEQ ID NO:1-10. Forexample, a fragment of SEQ ID NO:1-10 is useful as an immunogenicpeptide for the development of antibodies that specifically recognizeSEQ ID NO:1-10. The precise length of a fragment of SEQ ID NO:1-10 andthe region of SEQ ID NO:1-10 to which the fragment corresponds areroutinely determinable by one of ordinary skill in the art based on theintended purpose for the fragment.

A “full length” polynucleotide sequence is one containing at least atranslation initiation codon (e.g., methionine) followed by an openreading frame and a translation termination codon. A “full length”polynucleotide sequence encodes a “full length” polypeptide sequence.

“Homology” refers to sequence similarity or, interchangeably, sequenceidentity, between two or more polynucleotide sequences or two or morepolypeptide sequences.

The terms “percent identity” and “% identity,” as applied topolynucleotide sequences, refer to the percentage of residue matchesbetween at least two polynucleotide sequences aligned using astandardized algorithm. Such an algorithm may insert, in a standardizedand reproducible way, gaps in the sequences being compared in order tooptimize alignment between two sequences, and therefore achieve a moremeaningful comparison of the two sequences.

Percent identity between polynucleotide sequences may be determinedusing the default parameters of the CLUSTAL V algorithm as incorporatedinto the MEGALIGN version 3.12e sequence alignment program. This programis part of the LASERGENE software package, a suite of molecularbiological analysis programs (DNASTAR, Madison Wis.). CLUSTAL V isdescribed in Higgins, D. G. and P. M. Sharp (1989) CABIOS 5:151-153 andin Higgins, D. G. et al. (1992) CABIOS 8:189-191. For pairwisealignments of polynucleotide sequences, the default parameters are setas follows: Ktuple=2, gap penalty=5, window=4, and “diagonals saved”=4.The “weighted” residue weight table is selected as the default. Percentidentity is reported by CLUSTAL V as the “percent similarity” betweenaligned polynucleotide sequences.

Alternatively, a suite of commonly used and freely available sequencecomparison algorithms is provided by the National Center forBiotechnology Information (NCBI) Basic Local Alignment Search Tool(BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403-410), whichis available from several sources, including the NCBI, Bethesda, Md.,and on the Internet at http://www.ncbi.nlm.nih.gov/BLAST/. The BLASTsoftware suite includes various sequence analysis programs including“blastn,” that is used to align a known polynucleotide sequence withother polynucleotide sequences from a variety of databases. Alsoavailable is a tool called “BLAST 2 Sequences” that is used for directpairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” canbe accessed and used interactively athttp://www.ncbi.nlm.nih.gov/gorf/b12.html. The “BLAST 2 Sequences” toolcan be used for both blastn and blastp (discussed below). BLAST programsare commonly used with gap and other parameters set to default settings.For example, to compare two nucleotide sequences, one may use blastnwith the “BLAST 2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) set atdefault parameters. Such default parameters may be, for example:

Matrix: BLOSUM62

Reward for match: 1

Penalty for mismatch: −2

Open Gap: 5 and Extension Gap: 2 penalties

Gap x drop-off: 50

Expect: 10

Word Size: 11

Filter: on

Percent identity may be measured over the length of an entire definedsequence, for example, as defined by a particular SEQ ID number, or maybe measured over a shorter length, for example, over the length of afragment taken from a larger, defined sequence, for instance, a fragmentof at least 20, at least 30, at least 40, at least 50, at least 70, atleast 100, or at least 200 contiguous nucleotides. Such lengths areexemplary only, and it is understood that any fragment length supportedby the sequences shown herein, in the tables, figures, or SequenceListing, may be used to describe a length over which percentage identitymay be measured.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences due to the degeneracyof the genetic code. It is understood that changes in a nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid sequences that all encode substantially the same protein.

The phrases “percent identity” and “% identity,” as applied topolypeptide sequences, refer to the percentage of residue matchesbetween at least two polypeptide sequences aligned using a standardizedalgorithm. Methods of polypeptide sequence alignment are well-known.Some alignment methods take into account conservative amino acidsubstitutions. Such conservative substitutions, explained in more detailabove, generally preserve the charge and_hydrophobicity at the site ofsubstitution, thus preserving the structure (and therefore function) ofthe polypeptide.

Percent identity between polypeptide sequences may be determined usingthe default parameters of the CLUSTAL V algorithm as incorporated intothe MEGALIGN version 3.12e sequence alignment program (described andreferenced above). For pairwise alignments of polypeptide sequencesusing CLUSTAL V, the default parameters are set as follows: Ktuple=1,gap penalty=3, window=5, and “diagonals saved”=5. The PAM250 matrix isselected as the default residue weight table. As with polynucleotidealignments, the percent identity is reported by CLUSTAL V as the“percent similarity” between aligned polypeptide sequence pairs.

Alternatively the NCBI BLAST software suite may be used. For example,for a pairwise comparison of two polypeptide sequences, one may use the“BLAST 2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) with blastp setat default parameters. Such default parameters may be, for example:

Matrix: BLOSUM62

Open Gap: 11 and Extension Gap: 1 penalties

Gap x drop-off: 50

Expect: 10

Word Size: 3

Filter: on

Percent identity may be measured over the length of an entire definedpolypeptide sequence, for example, as defined by a particular SEQ IDnumber, or may be measured over a shorter length, for example, over thelength of a fragment taken from a larger, defined polypeptide sequence,for instance, a fragment of at least 15, at least 20, at least 30, atleast 40, at least 50, at least 70 or at least 150 contiguous residues.Such lengths are exemplary only, and it is understood that any fragmentlength supported by the sequences shown herein, in the tables, figuresor Sequence Listing, may be used to describe a length over whichpercentage identity may be measured.

“Human artificial chromosomes” (HACs) are linear microchromosomes whichmay contain DNA sequences of about 6 kb to 10 Mb in size and whichcontain all of the elements required for chromosome replication,segregation and maintenance.

The term “humanized antibody” refers to an antibody molecule in whichthe amino acid sequence in the non-antigen binding regions has beenaltered so that the antibody more closely resembles a human antibody,and still retains its original binding ability.

“Hybridization” refers to the process by which a polynucleotide strandanneals with a complementary strand through base pairing under definedhybridization conditions. Specific hybridization is an indication thattwo nucleic acid sequences share a high degree of complementarity.Specific hybridization complexes form under permissive annealingconditions and remain hybridized after the “washing” step(s). Thewashing step(s) is particularly important in determining the stringencyof the hybridization process, with more stringent conditions allowingless non-specific binding, i.e., binding between pairs of nucleic acidstrands that are not perfectly matched. Permissive conditions forannealing of nucleic acid sequences are routinely determinable by one ofordinary skill in the art and may be consistent among hybridizationexperiments, whereas wash conditions may be varied among experiments toachieve the desired stringency, and therefore hybridization specificity.Permissive annealing conditions occur, for example, at 68° C. in thepresence of about 6×SSC, about 1% (w/v) SDS, and about 100 μg/mlsheared, denatured salmon sperm DNA.

Generally, stringency of hybridization is expressed, in part, withreference to the temperature under which the wash step is carried out.Such wash temperatures are typically selected to be about 5° C. to 20°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. An equation forcalculating T_(m) and conditions for nucleic acid hybridization are wellknown and can be found in Sambrook, J. et al. (1989) Molecular Cloning:A Laboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor Press,Plainview N.Y.; specifically see volume 2, chapter 9.

High stringency conditions for hybridization between polynucleotides ofthe present invention include wash conditions of 68° C. in the presenceof about 0.2×SSC and about 0.1% SDS, for 1 hour. Alternatively,temperatures of about 65° C., 60° C., 55° C., or 42° C. may be used. SSCconcentration may be varied from about 0.1 to 2×SSC, with SDS beingpresent at about 0.1%. Typically, blocking reagents are used to blocknon-specific hybridization. Such blocking reagents include, forinstance, sheared and denatured salmon sperm DNA at about 100-200 μg/ml.Organic solvent, such as formamide at a concentration of about 35-50%v/v, may also be used under particular circumstances, such as forRNA:DNA hybridizations. Useful variations on these wash conditions willbe readily apparent to those of ordinary skill in the art.Hybridization, particularly under high stringency conditions, may besuggestive of evolutionary similarity between the nucleotides. Suchsimilarity is strongly indicative of a similar role for the nucleotidesand their encoded polypeptides.

The term “hybridization complex” refers to a complex formed between twonucleic acid sequences by virtue of the formation of hydrogen bondsbetween complementary bases. A hybridization complex may be formed insolution (e.g., C₀t or R₀t analysis) or formed between one nucleic acidsequence present in solution and another nucleic acid sequenceimmobilized on a solid support (e.g., paper, membranes, filters, chips,pins or glass slides, or any other appropriate substrate to which cellsor their nucleic acids have been fixed).

The words “insertion” and “addition” refer to changes in an amino acidor nucleotide sequence resulting in the addition of one or more aminoacid residues or nucleotides, respectively.

“Immune response” can refer to conditions associated with inflammation,trauma, immune disorders, or infectious or genetic disease, etc. Theseconditions can be characterized by expression of various factors, e.g.,cytokines, chemokines, and other signaling molecules, which may affectcellular and systemic defense systems.

An “immunogenic fragment” is a polypeptide or oligopeptide fragment ofGCREC which is capable of eliciting an immune response when introducedinto a living organism, for example, a mammal. The term “immunogenicfragment” also includes any polypeptide or oligopeptide fragment ofGCREC which is useful in any of the antibody production methodsdisclosed herein or known in the art.

The term “microarray” refers to an arrangement of a plurality ofpolynucleotides, polypeptides, or other chemical compounds on asubstrate.

The terms “element” and “array element” refer to a polynucleotide,polypeptide, or other chemical compound having a unique and definedposition on a microarray.

The term “modulate” refers to a change in the activity of GCREC. Forexample, modulation may cause an increase or a decrease in proteinactivity, binding characteristics, or any other biological, functional,or immunological properties of GCREC.

The phrases “nucleic acid” and “nucleic acid sequence” refer to anucleotide, oligonucleotide, polynucleotide, or any fragment thereof.These phrases also refer to DNA or RNA of genomic or synthetic originwhich may be single-stranded or double-stranded and may represent thesense or the antisense strand, to peptide nucleic acid (PNA), or to anyDNA-like or RNA-like material.

“Operably linked” refers to the situation in which a first nucleic acidsequence is placed in a functional relationship with a second nucleicacid sequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Operably linked DNA sequences may be in close proximityor contiguous and, where necessary to join two protein coding regions,in the same reading frame.

“Peptide nucleic acid” (PNA) refers to an antisense molecule oranti-gene agent which comprises an oligonucleotide of at least about 5nucleotides in length linked to a peptide backbone of amino acidresidues ending in lysine. The terminal lysine confers solubility to thecomposition. PNAs preferentially bind complementary single stranded DNAor RNA and stop transcript elongation, and may be pegylated to extendtheir lifespan in the cell.

“Post-translational modification” of an GCREC may involve lipidation,glycosylation, phosphorylation, acetylation, racemization, proteolyticcleavage, and other modifications known in the art. These processes mayoccur synthetically or biochemically. Biochemical modifications willvary by cell type depending on the enzymatic milieu of GCREC.

“Probe” refers to nucleic acid sequences encoding GCREC, theircomplements, or fragments thereof, which are used to detect identical,allelic or related nucleic acid sequences. Probes are isolatedoligonucleotides or polynucleotides attached to a detectable label orreporter molecule. Typical labels include radioactive isotopes, ligands,chemiluminescent agents, and enzymes. “Primers” are short nucleic acids,usually DNA oligonucleotides, which may be annealed to a targetpolynucleotide by complementary base-pairing. The primer may then beextended along the target DNA strand by a DNA polymerase enzyme. Primerpairs can be used for amplification (and identification) of a nucleicacid sequence, e.g., by the polymerase chain reaction (PCR).

Probes and primers as used in the present invention typically compriseat least 15 contiguous nucleotides of a known sequence. In order toenhance specificity, longer probes and primers may also be employed,such as probes and primers that comprise at least 20, 25, 30, 40, 50,60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of thedisclosed nucleic acid sequences. Probes and primers may be considerablylonger than these examples, and it is understood that any lengthsupported by the specification, including the tables, figures, andSequence Listing, may be used.

Methods for preparing and using probes and primers are described in thereferences, for example Sambrook, J. et al. (1989) Molecular Cloning: ALaboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor Press,Plainview N.Y.; Ausubel, F. M. et al. (1987) Current Protocols inMolecular Biology, Greene Publ. Assoc. & Wiley-Intersciences, New YorkN.Y.; Innis, M. et al. (1990) PCR Protocols. A Guide to Methods andApplications, Academic Press, San Diego Calif. PCR primer pairs can bederived from a known sequence, for example, by using computer programsintended for that purpose such as Primer (Version 0.5, 1991, WhiteheadInstitute for Biomedical Research, Cambridge Mass.).

Oligonucleotides for use as primers are selected using software known inthe art for such purpose. For example, OLIGO 4.06 software is useful forthe selection of PCR primer pairs of up to 100 nucleotides each, and forthe analysis of oligonucleotides and larger polynucleotides of up to5,000 nucleotides from an input polynucleotide sequence of up to 32kilobases. Similar primer selection programs have incorporatedadditional features for expanded capabilities. For example, the PrimOUprimer selection program (available to the public from the Genome Centerat University of Texas South West Medical Center, Dallas Tex.) iscapable of choosing specific primers from megabase sequences and is thususeful for designing primers on a genome-wide scope. The Primer3 primerselection program (available to the public from the WhiteheadInstitute/MIT Center for Genome Research, Cambridge Mass.) allows theuser to input a “mispriming library,” in which sequences to avoid asprimer binding sites are user-specified. Primer3 is useful, inparticular, for the selection of oligonucleotides for microarrays. (Thesource code for the latter two primer selection programs may also beobtained from their respective sources and modified to meet the user'sspecific needs.) The PrimeGen program (available to the public from theUK Human Genome Mapping Project Resource Centre, Cambridge UK) designsprimers based on multiple sequence alignments, thereby allowingselection of primers that hybridize to either the most conserved orleast conserved regions of aligned nucleic acid sequences. Hence, thisprogram is useful for identification of both unique and conservedoligonucleotides and polynucleotide fragments. The oligonucleotides andpolynucleotide fragments identified by any of the above selectionmethods are useful in hybridization technologies, for example, as PCR orsequencing primers, microarray elements, or specific probes to identifyfully or partially complementary polynucleotides in a sample of nucleicacids. Methods of oligonucleotide selection are not limited to thosedescribed above.

A “recombinant nucleic acid” is a sequence that is not naturallyoccurring or has a sequence that is made by an artificial combination oftwo or more otherwise separated segments of sequence. This artificialcombination is often accomplished by chemical synthesis or, morecommonly, by the artificial manipulation of isolated segments of nucleicacids, e.g., by genetic engineering techniques such as those describedin Sambrook, supra. The term recombinant includes nucleic acids thathave been altered solely by addition, substitution, or deletion of aportion of the nucleic acid. Frequently, a recombinant nucleic acid mayinclude a nucleic acid sequence operably linked to a promoter sequence.Such a recombinant nucleic acid may be part of a vector that is used,for example, to transform a cell.

Alternatively, such recombinant nucleic acids may be part of a viralvector, e.g., based on a vaccinia virus, that could be use to vaccinatea mammal wherein the recombinant nucleic acid is expressed, inducing aprotective immunological response in the mammal.

A “regulatory element” refers to a nucleic acid sequence usually derivedfrom untranslated regions of a gene and includes enhancers, promoters,introns, and 5′ and 3′ untranslated regions (UTRs). Regulatory elementsinteract with host or viral proteins which control transcription,translation, or RNA stability.

“Reporter molecules” are chemical or biochemical moieties used forlabeling a nucleic acid, amino acid, or antibody. Reporter moleculesinclude radionuclides; enzymes; fluorescent, chemiluminescent, orchromogenic agents; substrates; cofactors; inhibitors; magneticparticles; and other moieties known in the art.

An “RNA equivalent,” in reference to a DNA sequence, is composed of thesame linear sequence of nucleotides as the reference DNA sequence withthe exception that all occurrences of the nitrogenous base thymine arereplaced with uracil, and the sugar backbone is composed of riboseinstead of deoxyribose.

The term “sample” is used in its broadest sense. A sample suspected ofcontaining GCREC, nucleic acids encoding GCREC, or fragments thereof maycomprise a bodily fluid; an extract from a cell, chromosome, organelle,or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, insolution or bound to a substrate; a tissue; a tissue print; etc.

The terms “specific binding” and “specifically binding” refer to thatinteraction between a protein or peptide and an agonist, an antibody, anantagonist, a small molecule, or any natural or synthetic bindingcomposition. The interaction is dependent upon the presence of aparticular structure of the protein, e.g., the antigenic determinant orepitope, recognized by the binding molecule. For example, if an antibodyis specific for epitope “A,” the presence of a polypeptide comprisingthe epitope A, or the presence of free unlabeled A, in a reactioncontaining free labeled A and the antibody will reduce the amount oflabeled A that binds to the antibody.

The term “substantially purified” refers to nucleic acid or amino acidsequences that are removed from their natural environment and areisolated or separated, and are at least 60% free, preferably at least75% free, and most preferably at least 90% free from other componentswith which they are naturally associated.

A “substitution” refers to the replacement of one or more amino acidresidues or nucleotides by different amino acid residues or nucleotides,respectively.

“Substrate” refers to any suitable rigid or semi-rigid support includingmembranes, filters, chips, slides, wafers, fibers, magnetic ornonmagnetic beads, gels, tubing, plates, polymers, microparticles andcapillaries. The substrate can have a variety of surface forms, such aswells, trenches, pins, channels and pores, to which polynucleotides orpolypeptides are bound.

A “transcript image” refers to the collective pattern of gene expressionby a particular cell type or tissue under given conditions at a giventime.

“Transformation” describes a process by which exogenous DNA isintroduced into a recipient cell. Transformation may occur under naturalor artificial conditions according to various methods well known in theart, and may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod for transformation is selected based on the type of host cellbeing transformed and may include, but is not limited to, bacteriophageor viral infection, electroporation, heat shock, lipofection, andparticle bombardment. The term “transformed cells” includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, as well as transiently transformed cells which express theinserted DNA or RNA for limited periods of time.

A “transgenic organism,” as used herein, is any organism, including butnot limited to animals and plants, in which one or more of the cells ofthe organism contains heterologous nucleic acid introduced by way ofhuman intervention, such as by transgenic techniques well known in theart. The nucleic acid is introduced into the cell, directly orindirectly by introduction into a precursor of the cell, by way ofdeliberate genetic manipulation, such as by microinjection or byinfection with a recombinant virus. The term genetic manipulation doesnot include classical cross-breeding, or in vitro fertilization, butrather is directed to the introduction of a recombinant DNA molecule.The transgenic organisms contemplated in accordance with the presentinvention include bacteria, cyanobacteria, fungi, plants and animals.The isolated DNA of the present invention can be introduced into thehost by methods known in the art, for example infection, transfection,transformation or transconjugation. Techniques for transferring the DNAof the present invention into such organisms are widely known andprovided in references such as Sambrook et al. (1989), supra.

A “variant” of a particular nucleic acid sequence is defined as anucleic acid sequence having at least 40% sequence identity to theparticular nucleic acid sequence over a certain length of one of thenucleic acid sequences using blastn with the “BLAST 2 Sequences” toolVersion 2.0.9 (May 7, 1999) set at default parameters. Such a pair ofnucleic acids may show, for example, at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% or greater sequence identityover a certain defined length. A variant may be described as, forexample, an “allelic” (as defined above), “splice,” “species,” or“polymorphic” variant. A splice variant may have significant identity toa reference molecule, but will generally have a greater or lesser numberof polynucleotides due to alternative splicing of exons during mRNAprocessing. The corresponding polypeptide may possess additionalfunctional domains or lack domains that are present in the referencemolecule. Species variants are polynucleotide sequences that vary fromone species to another. The resulting polypeptides will generally havesignificant amino acid identity relative to each other. A polymorphicvariant is a variation in the polynucleotide sequence of a particulargene between individuals of a given species. Polymorphic variants alsomay encompass “single nucleotide polymorphisms” (SNPs) in which thepolynucleotide sequence varies by one nucleotide base. The presence ofSNPs may be indicative of, for example, a certain population, a diseasestate, or a propensity for a disease state.

A “variant” of a particular polypeptide sequence is defined as apolypeptide sequence having at least 40% sequence identity to theparticular polypeptide sequence over a certain length of one of thepolypeptide sequences using blastp with the “BLAST 2 Sequences” toolVersion 2.0.9 (May 7, 1999) set at default parameters. Such a pair ofpolypeptides may show, for example, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% or greater sequence identity over a certain definedlength of one of the polypeptides.

THE INVENTION

The invention is based on the discovery of new human G-protein coupledreceptors (GCREC), the polynucleotides encoding GCREC, and the use ofthese compositions for the diagnosis, treatment, or prevention of cellproliferative, neurological, cardiovascular, gastrointestinal,autoimrnune/inflammnatory, and metabolic disorders, and viralinfections.

Table 1 summarizes the nomenclature for the full length polynucleotideand polypeptide sequences of the invention. Each polynucleotide and itscorresponding polypeptide are correlated to a single Incyte projectidentification number (Incyte Project ID). Each polypeptide sequence isdenoted by both a polypeptide sequence identification number(Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number(Incyte Polypeptide ID) as shown. Each polynucleotide sequence isdenoted by both a polynucleotide sequence identification number(Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensussequence number (Incyte Polynucleotide ID) as shown.

Table 2 shows sequences with homology to the polypeptides of theinvention as identified by BLAST analysis against the GenBank protein(genpept) database. Columns 1 and 2 show the polypeptide sequenceidentification number (Polypeptide SEQ ID NO:) and the correspondingIncyte polypeptide sequence number (Incyte Polypeptide ID) forpolypeptides of the invention. Column 3 shows the GenBank identificationnumber (Genbank ID NO:) of the nearest GenBank homolog. Column 4 showsthe probability score for the match between each polypeptide and itsGenBank homolog. Column 5 shows the annotation of the GenBank homologalong with relevant citations where applicable, all of which areexpressly incorporated by reference herein.

Table 3 shows various structural features of the polypeptides of theinvention. Columns 1 and 2 show the polypeptide sequence identificationnumber (SEQ ID NO:) and the corresponding Incyte polypeptide sequencenumber (Incyte Polypeptide ID) for each polypeptide of the invention.Column 3 shows the number of amino acid residues in each polypeptide.Column 4 shows potential phosphorylation sites, and column 5 showspotential glycosylation sites, as determined by the MOTIFS program ofthe GCG sequence analysis software package (Genetics Computer Group,Madison Wis.). Column 6 shows amino acid residues comprising signaturesequences, domains, and motifs. Column 7 shows analytical methods forprotein structure/function analysis and in some cases, searchabledatabases to which the analytical methods were applied.

Together, Tables 2 and 3 summarize the properties of polypeptides of theinvention, and these properties establish that the claimed polypeptidesare G-protein coupled receptors. For example, SEQ ID NO:4 is 44%identical to murine olfactory receptor P2 (GenBank ID g7638409) asdetermined by the Basic Local Alignment Search Tool (BLAST). (See Table2.) The BLAST probability score is 4.6e-67, which indicates theprobability of obtaining the observed polypeptide sequence alignment bychance. SEQ ID NO:4 also contains a seven transmembrane receptor domainas determined by searching for statistically significant matches in thehidden Markov model (HMM)-based PFAM database of conserved proteinfamily domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCANanalyses provide further corroborative evidence that SEQ ID NO:4 is anolfactory G-protein coupled receptor. In an alternative example, SEQ IDNO:5 is 47% identical to murine olfactory receptor P2 (GenBank IDg7638409) as determined by BLAST. (See Table 2.) The BLAST probabilityscore is 2.2e-67. SEQ ID NO:5 also contains G-protein coupled receptorsignature domains as determined by searching for statisticallysignificant matches in the HMM-based PFAM database of conserved proteinfamily domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCANanalyses provide further corroborative evidence that SEQ ID NO:5 is aG-protein coupled receptor. In an alternative example, SEQ ID NO:7 is87% identical to mouse odorant receptor S1 (GenBank ID g4680254) asdetermined by BLAST. (See Table 2.) The BLAST probability score is1.1e-145. SEQ ID NO:7 also contains a 7 transmembrane receptor domaincharacteristic of the rhodopsin family, as determined by searching forstatistically significant matches in the HMM-based PFAM database ofconserved protein family domains. (See Table 3.) Data from BLIMPS,MOTIFS, and PROFILESCAN analyses provide further corroborative evidencethat SEQ ID NO:7 is a G-protein coupled receptor. SEQ ID NO:1-3, SEQ IDNO:6, and SEQ ID NO:8-10 were analyzed and annotated in a similarmanner. The algorithms and parameters for the analysis of SEQ ID NO:1-10are described in Table 7.

As shown in Table 4, the full length polynucleotide sequences of thepresent invention were assembled using cDNA sequences or coding (exon)sequences derived from genomic DNA, or any combination of these twotypes of sequences. Columns 1 and 2 list the polynucleotide sequenceidentification number (Polynucleotide SEQ ID NO:) and the correspondingIncyte polynucleotide consensus sequence number (Incyte PolynucleotideID) for each polynucleotide of the invention. Column 3 shows the lengthof each polynucleotide sequence in basepairs. Column 4 lists fragmentsof the polynucleotide sequences which are useful, for example, inhybridization or amplification technologies that identify SEQ IDNO:11-20 or that distinguish between SEQ ID NO:11-20 and relatedpolynucleotide sequences. Column 5 shows identification numberscorresponding to cDNA sequences, coding sequences (exons) predicted fromgenomic DNA, and/or sequence assemblages comprised of both cDNA andgenomic DNA. These sequences were used to assemble the full lengthpolynucleotide sequences of the invention. Columns 6 and 7 of Table 4show the nucleotide start (5′) and stop (3′) positions of the cDNAand/or genomic sequences in column 5 relative to their respective fulllength sequences.

The identification numbers in Column 5 of Table 4 may referspecifically, for example, to Incyte cDNAs along with theircorresponding cDNA libraries. For example, 5805033T6 is theidentification number of an Incyte cDNA sequence, and BONRFET03 is thecDNA library from which it is derived. Incyte cDNAs for which cDNAlibraries are not indicated were derived from pooled cDNA libraries(e.g., 55012833H1). Alternatively, the identification numbers in column5 may refer to GenBank cDNAs or ESTs which contributed to the assemblyof the full length polynucleotide sequences. Alternatively, theidentification numbers in column 5 may refer to coding regions predictedby Genscan analysis of genomic DNA. For example,GNN.g7283250_(—)000011_(—)008 is the identification number of aGenscan-predicted coding sequence, with g7283250 being the GenBankidentification number of the sequence to which Genscan was applied. TheGenscan-predicted coding sequences may have been edited prior toassembly. (See Example IV.) Alternatively, the identification numbers incolumn 5 may refer to assemblages of both cDNA and Genscan-predictedexons brought together by an “exon stitching” algorithm. For example,FL7472098CB1_(—)00001 represents a “stitched” sequence in which 7472098is the identification number of the cluster of sequences to which thealgorithm was applied, and 00001 is the number of the predictiongenerated by the algorithm. (See Example V.) Alternatively, theidentification numbers in column 5 may refer to assemblages of both cDNAand Genscan-predicted exons brought together by an “exon-stretching”algorithm. For example, FL7474927-g2822142-g4995709 is theidentification number of a “stretched” sequence, with 7474927 being theIncyte project identification number, g2822142 being the GenBankidentification number of the human genomic sequence to which the“exon-stretching” algorithm was applied, and g4995709 being the GenBankidentification number of the nearest GenBank protein homolog. (SeeExample V.) In some cases, Incyte cDNA coverage redundant with thesequence coverage shown in column 5 was obtained to confirm the finalconsensus polynucleotide sequence, but the relevant Incyte cDNAidentification numbers are not shown.

Table 5 shows the representative cDNA libraries for those full lengthpolynucleotide sequences which were assembled using Incyte cDNAsequences. The representative cDNA library is the Incyte cDNA librarywhich is most frequently represented by the Incyte cDNA sequences whichwere used to assemble and confirm the above polynucleotide sequences.The tissues and vectors which were used to construct the cDNA librariesshown in Table 5 are described in Table 6.

Table 8 shows tissue-specific expression of polynucleotides of theinvention. Column 1 lists groups of tissues which were tested bypolymerase chain reaction (PCR) for expression of the polynucleotides.The remaining columns indicate whether a particular polynucleotide wasexpressed in each tissue group. Detection of a PCR product indicatedpositive expression, denoted by a “+” sign, while inability to detect aPCR product indicated a lack of expression, denoted by a “−” sign.

The invention also encompasses GCREC variants. A preferred GCREC variantis one which has at least about 80%, or alternatively at least about90%, or even at least about 95% amino acid sequence identity to theGCREC amino acid sequence, and which contains at least one functional orstructural characteristic of GCREC.

The invention also encompasses polynucleotides which encode GCREC. In aparticular embodiment, the invention encompasses a polynucleotidesequence comprising a sequence selected from the group consisting of SEQID NO:11-20, which encodes GCREC. The polynucleotide sequences of SEQ IDNO:11-20, as presented in the Sequence Listing, embrace the equivalentRNA sequences, wherein occurrences of the nitrogenous base thymine arereplaced with uracil, and the sugar backbone is composed of riboseinstead of deoxyribose.

The invention also encompasses a variant of a polynucleotide sequenceencoding GCREC. In particular, such a variant polynucleotide sequencewill have at least about 70%, or alternatively at least about 85%, oreven at least about 95% polynucleotide sequence identity to thepolynucleotide sequence encoding GCREC. A particular aspect of theinvention encompasses a variant of a polynucleotide sequence comprisinga sequence selected from the group consisting of SEQ ID NO:11-20 whichhas at least about 70%, or alternatively at least about 85%, or even atleast about 95% polynucleotide sequence identity to a nucleic acidsequence selected from the group consisting of SEQ ID NO:11-20. Any oneof the polynucleotide variants described above can encode an amino acidsequence which contains at least one functional or structuralcharacteristic of GCREC.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of polynucleotidesequences encoding GCREC, some bearing minimal similarity to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequence of naturally occurring GCREC, and all suchvariations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode GCREC and its variants aregenerally capable of hybridizing to the nucleotide sequence of thenaturally occurring GCREC under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding GCREC or its derivatives possessing a substantially differentcodon usage, e.g., inclusion of non-naturally occurring codons. Codonsmay be selected to increase the rate at which expression of the peptideoccurs in a particular prokaryotic or eukaryotic host in accordance withthe frequency with which particular codons are utilized by the host.Other reasons for substantially altering the nucleotide sequenceencoding GCREC and its derivatives without altering the encoded aminoacid sequences include the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

The invention also encompasses production of DNA sequences which encodeGCREC and GCREC derivatives, or fragments thereof, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents well known in the art. Moreover, synthetic chemistry may beused to introduce mutations into a sequence encoding GCREC or anyfragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed polynucleotide sequences, and, inparticular, to those shown in SEQ ID NO:11-20 and fragments thereofunder various conditions of stringency. (See, e.g., Wahl, G. M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R. (1987)Methods Enzymol. 152:507-511.) Hybridization conditions, includingannealing and wash conditions, are described in “Definitions.”

Methods for DNA sequencing are well known in the art and may be used topractice any of the embodiments of the invention. The methods may employsuch enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (USBiochemical, Cleveland Ohio), Taq polymerase (Applied Biosystems),thermostable T7 polymerase (Amersham Pharmacia Biotech, PiscatawayN.J.), or combinations of polymerases and proofreading exonucleases suchas those found in the ELONGASE amplification system (Life Technologies,Gaithersburg Md.). Preferably, sequence preparation is automated withmachines such as the MICROLAB 2200 liquid transfer system (Hamilton,Reno Nev.), PTC200 thermal cycler (MJ Research, Watertown Mass.) and ABICATALYST 800 thermal cycler (Applied Biosystems). Sequencing is thencarried out using either the ABI 373 or 377 DNA sequencing system(Applied Biosystems), the MEGABACE 1000 DNA sequencing system (MolecularDynamics, Sunnyvale Calif.), or other systems known in the art. Theresulting sequences are analyzed using a variety of algorithms which arewell known in the art. (See, e.g., Ausubel, F. M. (1997) Short Protocolsin Molecular Biology, John Wiley & Sons, New York N.Y., unit 7.7;Meyers, R. A. (1995) Molecular Biology and Biotechnology, Wiley VCH, NewYork N.Y., pp. 856-853.)

The nucleic acid sequences encoding GCREC may be extended utilizing apartial nucleotide sequence and employing various PCR-based methodsknown in the art to detect upstream sequences, such as promoters andregulatory elements. For example, one method which may be employed,restriction-site PCR, uses universal and nested primers to amplifyunknown sequence from genomic DNA within a cloning vector. (See, e.g.,Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) Another method,inverse PCR, uses primers that extend in divergent directions to amplifyunknown sequence from a circularized template. The template is derivedfrom restriction fragments comprising a known genomic locus andsurrounding sequences. (See, e.g., Triglia, T. et al. (1988) NucleicAcids Res. 16:8186.) A third method, capture PCR, involves PCRamplification of DNA fragments adjacent to known sequences in human andyeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.(1991) PCR Methods Applic. 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to insert anengineered double-stranded sequence into a region of unknown sequencebefore performing PCR. Other methods which may be used to retrieveunknown sequences are known in the art. (See, e.g., Parker, J. D. et al.(1991) Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR,nested primers, and PROMOTERFINDER libraries (Clontech, Palo AltoCalif.) to walk genomic DNA. This procedure avoids the need to screenlibraries and is useful in finding intron/exon junctions. For allPCR-based methods, primers may be designed using commercially availablesoftware, such as OLIGO 4.06 primer analysis software (NationalBiosciences, Plymouth Minn.) or another appropriate program, to be about22 to 30 nucleotides in length, to have a GC content of about 50% ormore, and to anneal to the template at temperatures of about 68° C. to72° C.

When screening for full length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. In addition,random-primed libraries, which often include sequences containing the 5′regions of genes, are preferable for situations in which an oligo d(T)library does not yield a full-length cDNA. Genomic libraries may beuseful for extension of sequence into 5′ non-transcribed regulatoryregions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentnucleotide-specific, laser-stimulated fluorescent dyes, and a chargecoupled device camera for detection of the emitted wavelengths.Output/light intensity may be converted to electrical signal usingappropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, AppliedBiosystems), and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for sequencing smallDNA fragments which may be present in limited amounts in a particularsample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode GCREC may be cloned in recombinant DNAmolecules that direct expression of GCREC, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and used to express GCREC.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alterGCREC-encoding sequences for a variety of purposes including, but notlimited to, modification of the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example,oligonucleotide-mediated site-directed mutagenesis may be used tointroduce mutations that create new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, and so forth.

The nucleotides of the present invention may be subjected to DNAshuffling techniques such as MOLECULARBREEDING (Maxygen Inc., SantaClara Calif.; described in U.S. Pat. No. 5,837,458; Chang, C.-C. et al.(1999) Nat. Biotechnol. 17:793-797; Christians, F. C. et al. (1999) Nat.Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol.14:315-319) to alter or improve the biological properties of GCREC, suchas its biological or enzymatic activity or its ability to bind to othermolecules or compounds. DNA shuffling is a process by which a library ofgene variants is produced using PCR-mediated recombination of genefragments. The library is then subjected to selection or screeningprocedures that identify those gene variants with the desiredproperties. These preferred variants may then be pooled and furthersubjected to recursive rounds of DNA shuffling and selection/screening.Thus, genetic diversity is created through “artificial” breeding andrapid molecular evolution. For example, fragments of a single genecontaining random point mutations may be recombined, screened, and thenreshuffled until the desired properties are optimized. Alternatively,fragments of a given gene may be recombined with fragments of homologousgenes in the same gene family, either from the same or differentspecies, thereby maximizing the genetic diversity of multiple naturallyoccurring genes in a directed and controllable manner.

In another embodiment, sequences encoding GCREC may be synthesized, inwhole or in part, using chemical methods well known in the art. (See,e.g., Caruthers, M. H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223;and Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232.)Alternatively, GCREC itself or a fragment thereof may be synthesizedusing chemical methods. For example, peptide synthesis can be performedusing various solution-phase or solid-phase techniques. (See, e.g.,Creighton, T. (1984) Proteins, Structures and Molecular Properties, WHFreeman, New York N.Y., pp. 55-60; and Roberge, J. Y. et al. (1995)Science 269:202-204.) Automated synthesis may be achieved using the ABI431A peptide synthesizer (Applied Biosystems). Additionally, the aminoacid sequence of GCREC, or any part thereof, may be altered duringdirect synthesis and/or combined with sequences from other proteins, orany part thereof, to produce a variant polypeptide or a polypeptidehaving a sequence of a naturally occurring polypeptide.

The peptide may be substantially purified by preparative highperformance liquid chromatography. (See, e.g., Chiez, R. M. and F. Z.Regnier (1990) Methods Enzymol. 182:392-421.) The composition of thesynthetic peptides may be confirmed by amino acid analysis or bysequencing. (See, e.g., Creighton, supra, pp. 28-53.)

In order to express a biologically active GCREC, the nucleotidesequences encoding GCREC or derivatives thereof may be inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for transcriptional and translational control of theinserted coding sequence in a suitable host. These elements includeregulatory sequences, such as enhancers, constitutive and induciblepromoters, and 5′ and 3′ untranslated regions in the vector and inpolynucleotide sequences encoding GCREC. Such elements may vary in theirstrength and specificity. Specific initiation signals may also be usedto achieve more efficient translation of sequences encoding GCREC. Suchsignals include the ATG initiation codon and adjacent sequences, e.g.the Kozak sequence. In cases where sequences encoding GCREC and itsinitiation codon and upstream regulatory sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including an in-frame ATG initiation codonshould be provided by the vector. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate for the particular host cell system used. (See,e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding GCREC andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook, J.et al. (1989) Molecular Cloning. A Laboratory Manual, Cold Spring HarborPress, Plainview N.Y., ch. 4, 8, and 16-17; Ausubel, F. M. et al. (1995)Current Protocols in Molecular Biology, John Wiley & Sons, New YorkN.Y., ch. 9, 13, and 16.)

A variety of expression vector/host systems may be utilized to containand express sequences encoding GCREC. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); plant cell systemstransformed with viral expression vectors (e.g., cauliflower mosaicvirus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. (See,e.g., Sambrook, supra; Ausubel, supra; Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509; Engelhard, E. K. et al. (1994)Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum.Gene Ther. 7:1937-1945; Takamatsu, N. (1987) EMBO J. 6:307-311; TheMcGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, NewYork N.Y., pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad.Sci. USA 81:3655-3659; and Harrington, J. J. et al. (1997) Nat. Genet.15:345-355.) Expression vectors derived from retroviruses, adenoviruses,or herpes or vaccinia viruses, or from various bacterial plasmids, maybe used for delivery of nucleotide sequences to the targeted organ,tissue, or cell population. (See, e.g., Di Nicola, M. et al. (1998)Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc. Natl. Acad.Sci. USA 90(13):6340-6344; Buller, R. M. et al. (1985) Nature317(6040):813-815; McGregor, D. P. et al. (1994) Mol. Immunol.31(3):219-226; and Verma, I. M. and N. Somia (1997) Nature 389:239-242.)The invention is not limited by the host cell employed.

In bacterial systems, a number of cloning and expression vectors may beselected depending upon the use intended for polynucleotide sequencesencoding GCREC. For example, routine cloning, subdloning, andpropagation of polynucleotide sequences encoding GCREC can be achievedusing a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene,La Jolla Calif.) or PSPORT1 plasmid (Life Technologies). Ligation ofsequences encoding GCREC into the vector's multiple cloning sitedisrupts the lacZ gene, allowing a colorimetric screening procedure foridentification of transformed bacteria containing recombinant molecules.In addition, these vectors may be useful for in vitro transcription,dideoxy sequencing, single strand rescue with helper phage, and creationof nested deletions in the cloned sequence. (See, e.g., Van Heeke, G.and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When largequantities of GCREC are needed, e.g. for the production of antibodies,vectors which direct high level expression of GCREC may be used. Forexample, vectors containing the strong, inducible SP6 or T7bacteriophage promoter may be used.

Yeast expression systems may be used for production of GCREC. A numberof vectors containing constitutive or inducible promoters, such as alphafactor, alcohol oxidase, and PGH promoters, may be used in the yeastSaccharomyces cerevisiae or Pichia Rastoris. In addition, such vectorsdirect either the secretion or intracellular retention of expressedproteins and enable integration of foreign sequences into the hostgenome for stable propagation. (See, e.g., Ausubel, 1995, supra; Bitter,G. A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C. A. etal. (1994) Bio/Technology 12:181-184.)

Plant systems may also be used for expression of GCREC. Transcription ofsequences encoding GCREC may be driven by viral promoters, e.g., the 35Sand 19S promoters of CaMV used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984)EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; andWinter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. (See, e.g., The McGrawHill Yearbook of Science and Technology (1992) McGraw Hill, New YorkN.Y., pp. 191-196.)

In mammalian cells, a number of viral-based expression systems may beutilized. In cases where an adenovirus is used as an expression vector,sequences encoding GCREC may be ligated into an adenovimstranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain infective virus whichexpresses GCREC in host cells. (See, e.g., Logan, J. and T. Shenk (1984)Proc. Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcriptionenhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used toincrease expression in manmnalian host cells. SV40 or EBV-based vectorsmay also be used for high-level protein expression.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained in and expressed from aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes. (See, e.g., Harrington, J. J. etal. (1997) Nat. Genet. 15:345-355.)

For long term production of recombinant proteins in mammalian systems,stable expression of GCREC in cell lines is preferred. For example,sequences encoding GCREC can be transformed into cell lines usingexpression vectors which may contain viral origins of replication and/orendogenous expression elements and a selectable marker gene on the sameor on a separate vector. Following the introduction of the vector, cellsmay be allowed to grow for about 1 to 2 days in enriched media beforebeing switched to selective media. The purpose of the selectable markeris to confer resistance to a selective agent, and its presence allowsgrowth and recovery of cells which successfully express the introducedsequences. Resistant clones of stably transformed cells may bepropagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase and adenine phosphoribosyltransferase genes, for use intk and ape cells, respectively. (See, e.g., Wigler, M. et al. (1977)Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,antimetabolite, antibiotic, or herbicide resistance can be used as thebasis for selection. For example, dhfr confers resistance tomethotrexate; izeo confers resistance to the arninoglycosides neomycinand G-418; and als and pat confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M.et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin,F. et al. (1981) J. Mol. Biol. 150:1-14.) Additional selectable geneshave been described, e.g., trpB and hisD, which alter cellularrequirements for metabolites. (See, e.g., Hartan, S. C. and R. C.Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:8047-8051.) Visiblemarkers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech),β glucuronidase and its substrate β-glucuronide, or luciferase and itssubstrate luciferin may be used. These markers can be used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector systerl(See, e.g., Rhodes, C. A. (1995) Methods Mol. Biol. 55:121-131.)

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of thegene may need to be confirmed. For example, if the sequence encodingGCREC is inserted within a marker gene sequence, transformed cellscontaining sequences encoding GCREC can be identified by the absence ofmarker gene function. Alternatively, a marker gene can be placed intandem with a sequence encoding GCREC under the control of a singlepromoter. Expression of the marker gene in response to induction orselection usually indicates expression of the tandem gene as well.

In general, host cells that contain the nucleic acid sequence encodingGCREC and that express GCREC may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCRamplification, and protein bioassay or immunoassay techniques whichinclude membrane, solution, or chip based technologies for the detectionand/or quantification of nucleic acid or protein sequences.

Immunological methods for detecting and measuring the expression ofGCREC using either specific polyclonal or monoclonal antibodies areknown in the art. Examples of such techniques include enzyme-linkedimmnunosorbent assays (ELISAs), radioimmunoassays (RIAs), andfluorescence activated cell sorting (FACS). A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on GCREC is preferred, but a competitivebinding assay may be employed. These and other assays are well known inthe art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, aLaboratory Manual, APS Press, St. Paul Minn., Sect. IV; Coligan, J. E.et al. (1997) Current Protocols in Immunology, Greene Pub. Associatesand Wiley-Interscience, New York N.Y.; and Pound, J. D. (1998)Immunochemical Protocols, Humana Press, Totowa N.J.)

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding GCREC includeoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, the sequences encoding GCREC,or any fragments thereof, may be cloned into a vector for the productionof an mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits, such as those provided by AmershamPharmacia Biotech, Promega (Madison Wis.), and US Biochemical. Suitablereporter molecules or labels which may be used for ease of detectioninclude radionuclides, enzymes, fluorescent, chemiluminescent, orchromogenic agents, as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding GCREC may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or retained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeGCREC may be designed to contain signal sequences which direct secretionof GCREC through a prokaryotic or eukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” or “pro” form ofthe protein may also be used to specify protein targeting, folding,and/or activity. Different host cells which have specific cellularmachinery and characteristic mechanisms for post-translationalactivities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available fromthe American Type Culture Collection (ATCC, Manassas Va.) and may bechosen to ensure the correct modification and processing of the foreignprotein.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding GCREC may be ligated to aheterologous sequence resulting in translation of a fusion protein inany of the aforementioned host systems. For example, a chimeric GCRECprotein containing a heterologous moiety that can be recognized by acommercially available antibody may facilitate the screening of peptidelibraries for inhibitors of GCREC activity. Heterologous protein andpeptide moieties may also facilitate purification of fusion proteinsusing commercially available affinity matrices. Such moieties include,but are not limited to, glutathione S-transferase (GST), maltose bindingprotein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and6-His enable purification of their cognate fusion proteins onimmobilized glutathione, maltose, phenylarsine oxide, calmodulin, andmetal-chelate resins, respectively. FLAG, c-inyc, and hemagglutinin (HA)enable immunoaffinity purification of fusion proteins using commerciallyavailable monoclonal and polyclonal antibodies that specificallyrecognize these epitope tags. A fusion protein may also be engineered tocontain a proteolytic cleavage site located between the GCREC encodingsequence and the heterologous protein sequence, so that GCREC may becleaved away from the heterologous moiety following purification.Methods for fusion protein expression and purification are discussed inAusubel (1995, supra, ch. 10). A variety of commercially available kitsmay also be used to facilitate expression and purification of fusionproteins.

In a further embodiment of the invention, synthesis of radiolabeledGCREC may be achieved in vitro using the TNT rabbit reticulocyte lysateor wheat germ extract system (Promega). These systems coupletranscription and translation of protein-coding sequences operablyassociated with the T7, T3, or SP6 promoters. Translation takes place inthe presence of a radiolabeled amino acid precursor, for example,³⁵S-methionine.

GCREC of the present invention or fragments thereof may be used toscreen for compounds that specifically bind to GCREC. At least one andup to a plurality of test compounds may be screened for specific bindingto GCREC. Examples of test compounds include antibodies,oligonucleotides, proteins (e.g., receptors), or small molecules.

In one embodiment, the compound thus identified is closely related tothe natural ligand of GCREC, e.g., a ligand or fragment thereof, anatural substrate, a structural or functional mimetic, or a naturalbinding partner. (See, e.g., Coligan, J. E. et al. (1991) CurrentProtocols in Immunology 1(2): Chapter 5.) Similarly, the compound can beclosely related to the natural receptor to which GCREC binds, or to atleast a fragment of the receptor, e.g., the ligand binding site. Ineither case, the compound can be rationally designed using knowntechniques. In one embodiment, screening for these compounds involvesproducing appropriate cells which express GCREC, either as a secretedprotein or on the cell membrane. Preferred cells include cells frommammals, yeast, Drosophila, or E. coli. Cells expressing GCREC or cellmembrane fractions which contain GCREC are then contacted with a testcompound and binding, stimulation, or inhibition of activity of eitherGCREC or the compound is analyzed.

An assay may simply test binding of a test compound to the polypeptide,wherein binding is detected by a fluorophore, radioisotope, enzymeconjugate, or other detectable label. For example, the assay maycomprise the steps of combining at least one test compound with GCREC,either in solution or affixed to a solid support, and detecting thebinding of GCREC to the compound. Alternatively, the assay may detect ormeasure binding of a test compound in the presence of a labeledcompetitor. Additionally, the assay may be carried out using cell-freepreparations, chemical libraries, or natural product mixtures, and thetest compound(s) may be free in solution or affixed to a solid support.

GCREC of the present invention or fragments thereof may be used toscreen for compounds that modulate the activity of GCREC. Such compoundsmay include agonists, antagonists, or partial or inverse agonists. Inone embodiment, an assay is performed under conditions permissive forGCREC activity, wherein GCREC is combined with at least one testcompound, and the activity of GCREC in the presence of a test compoundis compared with the activity of GCREC in the absence of the testcompound. A change in the activity of GCREC in the presence of the testcompound is indicative of a compound that modulates the activity ofGCREC. Alternatively, a test compound is combined with an in vitro orcell-free system comprising GCREC under conditions suitable for GCRECactivity, and the assay is performed. In either of these assays, a testcompound which modulates the activity of GCREC may do so indirectly andneed not come in direct contact with the test compound. At least one andup to a plurality of test compounds may be screened.

In another embodiment, polynucleotides encoding GCREC or their mammalianhomologs may be “knocked out” in an animal model system using homologousrecombination in embryonic stem (ES) cells. Such techniques are wellknown in the art and are useful for the generation of animal models ofhuman disease. (See, e.g., U.S. Pat. No. 5,175,383 and U.S. Pat. No.5,767,337.) For example, mouse ES cells, such as the mouse 129/SvJ cellline, are derived from the early mouse embryo and grown in culture. TheES cells are transformed with a vector containing the gene of interestdisrupted by a marker gene, e.g., the neomycin phosphotransferase gene(neo; Capecchi, M. R. (1989) Science 244:1288-1292). The vectorintegrates into the corresponding region of the host genome byhomologous recombination. Alternatively, homologous recombination takesplace using the Cre-loxP system to knockout a gene of interest in atissue- or developmental stage-specific manner (Marth, J. D. (1996)Clin. Invest. 97:1999-2002; Wagner, K. U. et al. (1997) Nucleic AcidsRes. 25:4323-4330). Transformed ES cells are identified andmicroinjected into mouse cell blastocysts such as those from the C57BL/6mouse strain. The blastocysts are surgically transferred topseudopregnant dams, and the resulting chimeric progeny are genotypedand bred to produce heterozygous or homozygous strains. Transgenicanimals thus generated may be tested with potential therapeutic or toxicagents.

Polynucleotides encoding GCREC may also be manipulated in vitro in EScells derived from human blastocysts. Human ES cells have the potentialto differentiate into at least eight separate cell lineages includingendoderm, mesoderm, and ectodermal cell types. These cell lineagesdifferentiate into, for example, neural cells, hematopoietic lineages,and cardiomyocytes (Thomson, J. A. et al. (1998) Science 282:1145-1147).

Polynucleotides encoding GCREC can also be used to create “knockin”humanized animals (pigs) or transgenic animals (mice or rats) to modelhuman disease. With knockin technology, a region of a polynucleotideencoding GCREC is injected into animal ES cells, and the injectedsequence integrates into the animal cell genome. Transformed cells areinjected into blastulae, and the blastulae are implanted as describedabove. Transgenic progeny or inbred lines are studied and treated withpotential pharmaceutical agents to obtain information on treatment of ahuman disease. Alternatively, a mammal inbred to overexpress GCREC,e.g., by secreting GCREC in its milk may also serve as a convenientsource of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev.4:55-74).

Therapeutics

Chemical and structural similarity, e.g., in the context of sequencesand motifs, exists between regions of GCREC and G-protein coupledreceptors. In addition, the expression of GCREC is closely associatedwith bone and nasal tissue. In particular, the expression of SEQ IDNO:15 is associated with thymus tissue. Therefore, GCREC appears to playa role in cell proliferative, neurological, cardiovascular,gastrointestinal, autoimmune/inflammatory, and metabolic disorders, andviral infections. In the treatment of disorders associated withincreased GCREC expression or activity, it is desirable to decrease theexpression or activity of GCREC. In the treatment of disordersassociated with decreased GCREC expression or activity, it is desirableto increase the expression or activity of GCREC.

Therefore, in one embodiment, GCREC or a fragment or derivative thereofmay be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of GCREC. Examples ofsuch disorders include, but are not limited to, a cell proliferativedisorder such as actinic keratosis, arteriosclerosis, atherosclerosis,bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD),myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera,psoriasis, primary thrombocythemia, and cancers includingadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; a neurological disordersuch as epilepsy, ischemic cerebrovascular disease, stroke, cerebralneoplasms, Alzheimer's disease, Pick's disease, Huntington's disease,dementia, Parkinson's disease and other extrapyramidal disorders,amyotrophic lateral sclerosis and other motor neuron disorders,progressive neural muscular atrophy, retinitis pigmentosa, hereditaryataxias, multiple sclerosis and other demyelinating diseases, bacterialand viral meningitis, brain abscess, subdural empyema, epidural abscess,suppurative intracranial thrombophlebitis, myelitis and radiculitis,viral central nervous system disease, prion diseases including kuru,Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome,fatal familial insomnia, nutritional and metabolic diseases of thenervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinalhemangioblastomatosis, encephalotrigeminal syndrome, mental retardationand other developmental disorders of the central nervous system,cerebral palsy, neuroskeletal disorders, autonomic nervous systemdisorders, cranial nerve disorders, spinal cord diseases, musculardystrophy and other neuromuscular disorders, peripheral nervous systemdisorders, dermatomyositis and polymyositis, inherited, metabolic,endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis,mental disorders including mood, anxiety, and schizophrenic disorders,seasonal affective disorder (SAD), akathesia, amnesia, catatonia,diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses,postherpetic neuralgia, Tourette's disorder, progressive supranuclearpalsy, corticobasal degeneration, and familial frontotemporal dementia;a cardiovascular disorder such as arteriovenous fistula,atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms,arterial dissections, varicose veins, thrombophlebitis andphlebothrombosis, vascular tumors, complications of thrombolysis,balloon angioplasty, vascular replacement, and coronary artery bypassgraft surgery, congestive heart failure, ischemic heart disease, anginapectoris, myocardial infarction, hypertensive heart disease,degenerative valvular heart disease, calcific aortic valve stenosis,congenitally bicuspid aortic valve, mitral annular calcification, mitralvalve prolapse, rheumatic fever and rheumatic heart disease, infectiveendocarditis, nonbacterial thrombotic endocarditis, endocarditis ofsystemic lupus erythematosus, carcinoid heart disease, cardiomyopathy,myocarditis, pericarditis, neoplastic heart disease, congenital heartdisease, and complications of cardiac transplantation; agastrointestinal disorder such as dysphagia, peptic esophagitis,esophageal spasm, esophageal stricture, esophageal carcinoma, dyspepsia,indigestion, gastritis, gastric carcinoma, anorexia, nausea, emesis,gastroparesis, antral or pyloric edema, abdominal angina, pyrosis,gastroenteritis, intestinal obstruction, infections of the intestinaltract, peptic ulcer, cholelithiasis, cholecystitis, cholestasis,pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis,hyperbilirubinemia, cirrhosis, passive congestion of the liver,hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis,Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, coloniccarcinoma, colonic obstruction, irritable bowel syndrome, short bowelsyndrome, diarrhea, constipation, gastrointestinal hemorrhage, acquiredimmunodeficiency syndrome (AIDS) enteropathy, jaundice, hepaticencephalopathy, hepatorenal syndrome, hepatic steatosis,hemochromatosis, Wilson's disease, alpha₁-antitrypsin deficiency, Reye'ssyndrome, primary sclerosing cholangitis, liver infarction, portal veinobstruction and thrombosis, centrilobular necrosis, peliosis hepatis,hepatic vein thrombosis, veno-occlusive disease, preeclampsia,eclampsia, acute fatty liver of pregnancy, intrahepatic cholestasis ofpregnancy, and hepatic tumors including nodular hyperplasias, adenomas,and carcinomas; an autoimmune/inflammatory disorder such as acquiredimmunodeficiency syndrome (AIDS), Addison's disease, adult respiratorydistress syndrome, allergies, ankylosing spondylitis, amyloidosis,anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmunethyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermaldystrophy (APECED), bronchitis, cholecystitis, contact dermatitis,Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosisfetalis, erythema nodosum, atrophic gastritis, glomerulonephritis,Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis,hypereosinophilia, irritable bowel syndrome, multiple sclerosis,myasthenia gravis, myocardial or pericardial inflammation,osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis,Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjögren'ssyndrome, systemic anaphylaxis, systemic lupus erythematosus, systemicsclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Wernersyndrome, complications of cancer, hemodialysis, and extracorporealcirculation, viral, bacterial, fungal, parasitic, protozoal, andhelminthic infections, and trauma; a metabolic disorder such asdiabetes, obesity, and osteoporosis; and an infection by a viral agentclassified as adenovirus, arenavirus, bunyavirus, calicivirus,coronavirus, filovirus, hepadnavirus, herpesvirus, flavivirus,orthomyxovirus, parvovirus, papovavirus, paramyxovirus, picornavirus,poxvirus, reovirus, retrovirus, rhabdovirus, and tongavirus.

In another embodiment, a vector capable of expressing GCREC or afragment or derivative thereof may be administered to a subject to treator prevent a disorder associated with decreased expression or activityof GCREC including, but not limited to, those described above.

In a further embodiment, a composition comprising a substantiallypurified GCREC in conjunction with a suitable pharmaceutical carrier maybe administered to a subject to treat or prevent a disorder associatedwith decreased expression or activity of GCREC including, but notlimited to, those provided above.

In still another embodiment, an agonist which modulates the activity ofGCREC may be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of GCREC including, butnot limited to, those listed above.

In a further embodiment, an antagonist of GCREC may be administered to asubject to treat or prevent a disorder associated with increasedexpression or activity of GCREC. Examples of such disorders include, butare not limited to, those cell proliferative, neurological,cardiovascular, gastrointestinal, autoimmune/inflammatory, and metabolicdisorders, and viral infections, described above. In one aspect, anantibody which specifically binds GCREC may be used directly as anantagonist or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissues which express GCREC.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding GCREC may be administered to a subject to treator prevent a disorder associated with increased expression or activityof GCREC including, but not limited to, those described above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences, or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of GCREC may be produced using methods which are generallyknown in the art. In particular, purified GCREC may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind GCREC. Antibodies to GCREC may also begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,and single chain antibodies, Fab fragments, and fragments produced by aFab expression library. Neutralizing antibodies (i.e., those whichinhibit dimer formation) are generally preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith GCREC or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to GCREC have an amino acid sequence consisting of atleast about 5 amino acids, and generally will consist of at least about10 amino acids. It is also preferable that these oligopeptides,peptides, or fragments are identical to a portion of the amino acidsequence of the natural protein. Short stretches of GCREC amino acidsmay be fused with those of another protein, such as KLH, and antibodiesto the chimeric molecule may be produced.

Monoclonal antibodies to GCREC may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; and Cole, S. P. etal. (1984) Mol. Cell Biol. 62:109-120.)

In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature314:452-454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce GCREC-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries. (See, e.g., Burton, D. R. (1991) Proc. Natl. Acad. Sci. USA88:10134-10137.)

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature.(See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)

Antibody fragments which contain specific binding sites for GCREC mayalso be generated. For example, such fragments include, but are notlimited to, F(ab)₂ fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab′)2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (See, e.g., Huse,W. D. et al. (1989) Science 246:1275-1281.)

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between GCREC and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering GCREC epitopes is generally used, but a competitivebinding assay may also be employed (Pound, supra.

Various methods such as Scatchard analysis in conjunction withradioimmunoassay techniques may be used to assess the affinity ofantibodies for GCREC. Affinity is expressed as an association constant,K_(a), which is defined as the molar concentration of GCREC-antibodycomplex divided by the molar concentrations of free antigen and freeantibody under equilibrium conditions. The K_(a) determined for apreparation of polyclonal antibodies, which are heterogeneous in theiraffinities for multiple GCREC epitopes, represents the average affinity,or avidity, of the antibodies for GCREC. The K_(a) determined for apreparation of monoclonal antibodies, which are monospecific for aparticular GCREC epitope, represents a true measure of affinity.High-affinity antibody preparations with K_(a) ranging from about 10⁹ to10¹² L/mole are preferred for use in immunoassays in which theGCREC-antibody complex must withstand rigorous manipulations.Low-affinity antibody preparations with K_(a) ranging from about 10⁶ to10¹² L/mole are preferred for use in immunopurification and similarprocedures which ultimately require dissociation of GCREC, preferably inactive form, from the antibody (Catty, D. (1988) Antibodies, Volume I: APractical Approach, IRL Press, Washington DC; Liddell, J. E. and A.Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley &Sons, New York N.Y.).

The titer and avidity of polyclonal antibody preparations may be furtherevaluated to determine the quality and suitability of such preparationsfor certain downstream applications. For example, a polyclonal antibodypreparation containing at least 1-2 mg specific antibody/ml, preferably5-10 mg specific antibody/ml, is generally employed in proceduresrequiring precipitation of GCREC-antibody complexes. Procedures forevaluating antibody specificity, titer, and avidity, and guidelines forantibody quality and usage in various applications, are generallyavailable. (See, e.g., Catty, supra, and Coligan et al. supra.)

In another embodiment of the invention, the polynucleotides encodingGCREC, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, modifications of gene expressioncan be achieved by designing complementary sequences or antisensemolecules (DNA, RNA, PNA, or modified oligonucleotides) to the coding orregulatory regions of the gene encoding GCREC. Such technology is wellknown in the art, and antisense oligonucleotides or larger fragments canbe designed from various locations along the coding or control regionsof sequences encoding GCREC. (See, e.g., Agrawal, S., ed. (1996)Antisense Therapeutics, Humana Press Inc., Totawa N.J.)

In therapeutic use, any gene delivery system suitable for introductionof the antisense sequences into appropriate target cells can be used.Antisense sequences can be delivered intracellularly in the form of anexpression plasmid which, upon transcription, produces a sequencecomplementary to at least a portion of the cellular sequence encodingthe target protein. (See, e.g., Slater, J. E. et al. (1998) J. AllergyClin. Immunol. 102(3):469-475; and Scanlon, K. J. et al. (1995)9(13):1288-1296.) Antisense sequences can also be introducedintracellularly through the use of viral vectors, such as retrovirns andadeno-associated virus vectors. (See, e.g., Miller, A. D. (1990) Blood76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol.Ther. 63(3):323-347.) Other gene delivery mechanisms includeliposome-derived systems, artificial viral envelopes, and other systemsknown in the art. (See, e.g., Rossi, J. J. (1995) Br. Med. Bull.51(1):217-225; Boado, R. J. et al. (1998) J. Pharm. Sci.87(11):1308-1315; and Morris, M. C. et al. (1997) Nucleic Acids Res.25(14):2730-2736.)

In another embodiment of the invention, polynucleotides encoding GCRECmay be used for somatic or germline gene therapy. Gene therapy may beperformed to (i) correct a genetic deficiency (e.g., in the cases ofsevere combined immunodeficiency (SCID)-X1 disease characterized byX-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science288:669-672), severe combined immunodeficiency syndrome associated withan inherited adenosine deaminase (ADA) deficiency (Blaese, R. M. et al.(1995) Science 270:475-480; Bordignon, C. et al. (1995) Science270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216;Crystal, R. G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R. G.et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familialhypercholesterolemia, and hemophilia resulting from Factor VIII orFactor IX deficiencies (Crystal, R. G. (1995) Science 270:404-410;Verma, I. M. and N. Somia (1997) Nature 389:239-242)), (ii) express aconditionally lethal gene product (e.g., in the case of cancers whichresult from unregulated cell proliferation), or (iii) express a proteinwhich affords protection against intracellular parasites (e.g., againsthuman retroviruses, such as human immunodeficiency virus (HIV)(Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996)Proc. Natl. Acad. Sci. USA. 93:11395-11399), hepatitis B or C virus(HBV, HCV); fungal parasites, such as Candida albicans andParacoccidioides brasiliensis; and protozoan parasites such asPlasmodium falciparum and Trypanosoma cruzi). In the case where agenetic deficiency in GCREC expression or regulation causes disease, theexpression of GCREC from an appropriate population of transduced cellsmay alleviate the clinical manifestations caused by the geneticdeficiency.

In a further embodiment of the invention, diseases or disorders causedby deficiencies in GCREC are treated by constructing mammalianexpression vectors encoding GCREC and introducing these vectors bymechanical means into GCREC-deficient cells. Mechanical transfertechnologies for use with cells in vivo or ex vitro include (i) directDNA microinjection into individual cells, (ii) ballistic gold particledelivery, (iii) liposome-mediated transfection, (iv) receptor-mediatedgene transfer, and (v) the use of DNA transposons (Morgan, R. A. and W.F. Anderson (1993) Annu. Rev. Biochem. 62:191-217; Ivics, Z. (1997) Cell91:501-510; Boulay, J-L. and H. Recipon (1998) Curr. Opin. Biotechnol.9:445-450).

Expression vectors that may be effective for the expression of GCRECinclude, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP,PVAX vectors (Invitrogen, Carlsbad Calif.), PCMV-SCRIPT, PCMV-TAG,PEGSHIPERV (Stratagene, La Jolla Calif.), and PTET-OFF, PTET-ON, PTRE2,PTRE2-LUC, PTK-HYG (Clontech, Palo Alto Calif.). GCREC may be expressedusing (i) a constitutively active promoter, (e.g., from cytomegalovirus(CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), orβ-actin genes), (ii) an inducible promoter (e.g., thetetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc.Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science268:1766-1769; Rossi, F. M. V. and H. M. Blau (1998) Curr. Opin.Biotechnol. 9:451-456), commercially available in the T-REX plasmid(Invitrogen)); the ecdysone-inducible promoter (available in theplasmids PVGRXR and PIND; Invitrogen); the FK506/rapamycin induciblepromoter; or the RU486/mifepristone inducible promoter (Rossi, F. M. V.and Blau, H. M. supra)), or (iii) a tissue-specific promoter or thenative promoter of the endogenous gene encoding GCREC from a normalindividual.

Commercially available liposome transformation kits (e.g., the PERFECTLIPID TRANSFECTION KIT, available from Invitrogen) allow one withordinary skill in the art to deliver polynucleotides to target cells inculture and require minimal effort to optimize experimental parameters.In the alternative, transformation is performed using the calciumphosphate method (Graham, F. L. and A. J. Eb (1973) Virology52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J.1:841-845). The introduction of DNA to primary cells requiresmodification of these standardized mammalian transfection protocols.

In another embodiment of the invention, diseases or disorders caused bygenetic defects with respect to GCREC expression are treated byconstructing a retrovirus vector consisting of (i) the polynucleotideencoding GCREC under the control of an independent promoter or theretrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNApackaging signals, and (iii) a Rev-responsive element (RRE) along withadditional retrovirus cis-acting RNA sequences and coding sequencesrequired for efficient vector propagation. Retrovirus vectors (e.g., PFBand PFBNEO) are commercially available (Stratagene) and are based onpublished data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. USA92:6733-6737), incorporated by reference herein. The vector ispropagated in an appropriate vector producing cell line (VPCL) thatexpresses an envelope gene with a tropism for receptors on the targetcells or a promiscuous envelope protein such as VSVg (Armentano, D. etal. (1987) J. Virol. 61:1647-1650; Bender, M. A. et al. (1987) J. Virol.61:1639-1646; Adam, M. A. and A. D. Miller (1988) J. Virol.62:3802-3806; Dull, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey,R. et al. (1998) J. Virol. 72:9873-9880). U.S. Pat. No. 5,910,434 toRigg (“Method for obtaining retrovirus packaging cell lines producinghigh transducing efficiency retroviral supernatant”) discloses a methodfor obtaining retrovirus packaging cell lines and is hereby incorporatedby reference. Propagation of retrovirus vectors, transduction of apopulation of cells (e.g., CD4⁺ T-cells), and the return of transducedcells to a patient are procedures well known to persons skilled in theart of gene therapy and have been well documented (Ranga, U. et al.(1997) J. Virol. 71:7020-7029; Bauer, G. et al. (1997) Blood89:2259-2267; Bonyhadi, M. L. (1997) J. Virol. 71:4707-4716; Ranga, U.et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997)Blood 89:2283-2290).

In the alternative, an adenovirus-based gene therapy delivery system isused to deliver polynucleotides encoding GCREC to cells which have oneor more genetic abnormalities with respect to the expression of GCREC.The construction and packaging of adenovirus-based vectors are wellknown to those with ordinary skill in the art. Replication defectiveadenovirus vectors have proven to be versatile for importing genesencoding immunoregulatory proteins into intact islets in the pancreas(Csete, M. E. et al. (1995) Transplantation 27:263-268). Potentiallyuseful adenoviral vectors are described in U.S. Pat. No. 5,707,618 toArmentano (“Adenovirus vectors for gene therapy”), hereby incorporatedby reference. For adenoviral vectors, see also Antinozzi, P. A. et al.(1999) Annu. Rev. Nutr. 19:511-544 and Verma, I. M. and N. Somia (1997)Nature 18:389:239-242, both incorporated by reference herein.

In another alternative, a herpes-based, gene therapy delivery system isused to deliver polynucleotides encoding GCREC to target cells whichhave one or more genetic abnormalities with respect to the expression ofGCREC. The use of herpes simplex virus (HSV)-based vectors may beespecially valuable for introducing GCREC to cells of the centralnervous system, for which HSV has a tropism. The construction andpackaging of herpes-based vectors are well known to those with ordinaryskill in the art. A replication-competent herpes simplex virus (HSV)type 1-based vector has been used to deliver a reporter gene to the eyesof primates (Liu, X. et al. (1999) Exp. Eye Res. 169:385-395). Theconstruction of a HSV-1 virus vector has also been disclosed in detailin U.S. Pat. No. 5,804,413 to DeLuca (“Herpes simplex virus strains forgene transfer”), which is hereby incorporated by reference. U.S. Pat.No. 5,804,413 teaches the use of recombinant HSV d92 which consists of agenome containing at least one exogenous gene to be transferred to acell under the control of the appropriate promoter for purposesincluding human gene therapy. Also taught by this patent are theconstruction and use of recombinant HSV strains deleted for ICP4, ICP27and ICP22. For HSV vectors, see also Goins, W. F. et al. (1999) J.Virol. 73:519-532 and Xu, H. et al. (1994) Dev. Biol. 163:152-161,hereby incorporated by reference. The manipulation of cloned herpesvirussequences, the generation of recombinant virus following thetransfection of multiple plasmids containing different segments of thelarge herpesvirus genomes, the growth and propagation of herpesvirus,and the infection of cells with herpesvirus are techniques well known tothose of ordinary skill in the art.

In another alternative, an alphavirus (positive, single-stranded RNAvirus) vector is used to deliver polynucleotides encoding GCREC totarget cells. The biology of the prototypic alphavirus, Semliki ForestVirus (SFV), has been studied extensively and gene transfer vectors havebeen based on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin.Biotechnol. 9:464-469). During alphavirus RNA replication, a subgenomicRNA is generated that normally encodes the viral capsid proteins. Thissubgenomic RNA replicates to higher levels than the full length genomicRNA, resulting in the overproduction of capsid proteins relative to theviral proteins with enzymatic activity (e.g., protease and polymerase).Similarly, inserting the coding sequence for GCREC into the alphavirusgenome in place of the capsid-coding region results in the production ofa large number of GCREC-coding RNAs and the synthesis of high levels ofGCREC in vector transduced cells. While alphavirus infection istypically associated with cell lysis within a few days, the ability toestablish a persistent infection in hamster normal kidney cells (BHK-21)with a variant of Sindbis virus (SIN) indicates that the lyticreplication of alphaviruses can be altered to suit the needs of the genetherapy application (Dryga, S. A. et al. (1997) Virology 228:74-83). Thewide host range of alphaviruses will allow the introduction of GCRECinto a variety of cell types. The specific transduction of a subset ofcells in a population may require the sorting of cells prior totransduction. The methods of manipulating infectious cDNA clones ofalphaviruses, performing alphavirus cDNA and RNA transfections, andperforming alphavirus infections, are well known to those with ordinaryskill in the art.

Oligonucleotides derived from the transcription initiation site, e.g.,between about positions −10 and +10 from the start site, may also beemployed to inhibit gene expression. Similarly, inhibition can beachieved using triple helix base-pairing methodology. Triple helixpairing is useful because it causes inhibition of the ability of thedouble helix to open sufficiently for the binding of polymerases,transcription factors, or regulatory molecules. Recent therapeuticadvances using triplex DNA have been described in the literature. (See,e.g., Gee, J. E. et al. (1994) in Huber, B. E. and B. I. Carr, Molecularand Immununologic Approaches, Futura Publishing, Mt. Kisco N.Y., pp.163-177.) A complementary sequence or antisense molecule may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingGCREC.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding GCREC. SuchDNA sequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA, constitutivelyor inducibly, can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the molecule,or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

An additional embodiment of the invention encompasses a method forscreening for a compound which is effective in altering expression of apolynucleotide encoding GCREC. Compounds which may be effective inaltering expression of a specific polynucleotide may include, but arenot limited to, oligonucleotides, antisense oligonucleotides, triplehelix-forming oligonucleotides, transcription factors and otherpolypeptide transcriptional regulators, and non-macromolecular chemicalentities which are capable of interacting with specific polynucleotidesequences. Effective compounds may alter polynucleotide expression byacting as either inhibitors or promoters of polynucleotide expression.Thus, in the treatment of disorders associated with increased GCRECexpression or activity, a compound which specifically inhibitsexpression of the polynucleotide encoding GCREC may be therapeuticallyuseful, and in the treatment of disorders associated with decreasedGCREC expression or activity, a compound which specifically promotesexpression of the polynucleotide encoding GCREC may be therapeuticallyuseful.

At least one, and up to a plurality, of test compounds may be screenedfor effectiveness in altering expression of a specific polynucleotide. Atest compound may be obtained by any method commonly known in the art,including chemical modification of a compound known to be effective inaltering polynucleotide expression; selection from an existing,commercially-available or proprietary library of naturally-occurring ornon-natural chemical compounds; rational design of a compound based onchemical and/or structural properties of the, target polynucleotide; andselection from a library of chemical compounds created combinatoriallyor randomly. A sample comprising a polynucleotide encoding GCREC isexposed to at least one test compound thus obtained. The sample maycomprise, for example, an intact or permeabilized cell, or an in vitrocell-free or reconstituted biochemical system. Alterations in theexpression of a polynucleotide encoding GCREC are assayed by any methodcommonly known in the art. Typically, the expression of a specificnucleotide is detected by hybridization with a probe having a nucleotidesequence complementary to the sequence of the polynucleotide encodingGCREC. The amount of hybridization may be quantified, thus forming thebasis for a comparison of the expression of the polynucleotide both withand without exposure to one or more test compounds. Detection of achange in the expression of a polynucleotide exposed to a test compoundindicates that the test compound is effective in altering the expressionof the polynucleotide. A screen for a compound effective in alteringexpression of a specific polynucleotide can be carried out, for example,using a Schizosaccharomyces pombe gene expression system (Atkins, D. etal. (1999) U.S. Pat. No. 5,932,435; Arndt, G. M. et al. (2000) NucleicAcids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M. L.et al. (2000) Biochem. Biophys. Res. Commun. 268:8-13). A particularembodiment of the present invention involves screening a combinatoriallibrary of oligonucleotides (such as deoxyribonucleotides,ribonucleotides, peptide nucleic acids, and modified oligonucleotides)for antisense activity against a specific polynucleotide sequence(Bruice, T. W. et al. (1997) U.S. Pat. No. 5,686,242; Bruice, T. W. etal. (2000) U.S. Pat. No. 6,022,691).

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art. (See, e.g., Goldman, C. K. et al. (1997) Nat.Biotechnol. 15:462-466.)

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such ashumans, dogs, cats, cows, horses, rabbits, and monkeys.

An additional embodiment of the invention relates to the administrationof a composition which generally comprises an active ingredientformulated with a pharmaceutically acceptable excipient. Excipients mayinclude, for example, sugars, starches, celluloses, gums, and proteins.Various formulations are commonly known and are thoroughly discussed inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing, Easton Pa.). Such compositions may consist of GCREC,antibodies to GCREC, and mimetics, agonists, antagonists, or inhibitorsof GCREC.

The compositions utilized in this invention may be administered by anynumber of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal,intranasal, enteral, topical, sublingual, or rectal means.

Compositions for pulmonary administration may be prepared in liquid ordry powder form. These compositions are generally aerosolizedimmediately prior to inhalation by the patient. In the case of smallmolecules (e.g. traditional low molecular weight organic drugs), aerosoldelivery of fast-acting formulations is well-known in the art. In thecase of macromolecules (e.g. larger peptides and proteins), recentdevelopments in the field of pulmonary delivery via the alveolar regionof the lung have enabled the practical delivery of drugs such as insulinto blood circulation (see, e.g., Patton, J. S. et al., U.S. Pat. No.5,997,848). Pulmonary delivery has the advantage of administrationwithout needle injection, and obviates the need for potentially toxicpenetration enhancers.

Compositions suitable for use in the invention include compositionswherein the active ingredients are contained in an effective amount toachieve the intended purpose. The determination of an effective dose iswell within the capability of those skilled in the art.

Specialized forms of compositions may be prepared for directintracellular delivery of macromolecules comprising GCREC or fragmentsthereof. For example, liposome preparations containing acell-impermeable macromolecule may promote cell fusion and intracellulardelivery of the macromolecule. Alternatively, GCREC or a fragmentthereof may be joined to a short cationic N-terminal portion from theHIV Tat-1 protein. Fusion proteins thus generated have been found totransduce into the cells of all tissues, including the brain, in a mousemodel system (Schwarze, S. R. et al. (1999) Science 285:1569-1572).

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models such as mice, rats, rabbits, dogs, monkeys, or pigs. Ananimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example GCREC or fragments thereof, antibodies of GCREC,and agonists, antagonists or inhibitors of GCREC, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD₅₀ (the doselethal to 50% of the population) statistics. The dose ratio of toxic totherapeutic effects is the therapeutic index, which can be expressed asthe LD₅₀/ED₅₀ ratio. Compositions which exhibit large therapeuticindices are preferred. The data obtained from cell culture assays andanimal studies are used to formulate a range of dosage for human use.The dosage contained in such compositions is preferably within a rangeof circulating concentrations that includes the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, the sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting compositions may beadministered every 3 to 4 days, every week, or biweekly depending on thehalf-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to atotal dose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnositics

In another embodiment, antibodies which specifically bind GCREC may beused for the diagnosis of disorders characterized by expression ofGCREC, or in assays to monitor patients being treated with GCREC oragonists, antagonists, or inhibitors of GCREC. Antibodies useful fordiagnostic purposes may be prepared in the same manner as describedabove for therapeutics. Diagnostic assays for GCREC include methodswhich utilize the antibody and a label to detect GCREC in human bodyfluids or in extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by covalent ornon-covalent attachment of a reporter molecule. A wide variety ofreporter molecules, several of which are described above, are known inthe art and may be used.

A variety of protocols for measuring GCREC, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of GCREC expression. Normal or standard values for GCRECexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, for example, human subjects, withantibodies to GCREC under conditions suitable for complex formation. Theamount of standard complex formation may be quantitated by variousmethods, such as photometric means. Quantities of GCREC expressed insubject, control, and disease samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingGCREC may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantify gene expression in biopsied tissues in which expression ofGCREC may be correlated with disease. The diagnostic assay may be usedto determine absence, presence, and excess expression of GCREC, and tomonitor regulation of GCREC levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding GCREC or closely related molecules may be used to identifynucleic acid sequences which encode GCREC. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5′regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification willdetermine whether the probe identifies only naturally occurringsequences encoding GCREC, allelic variants, or related sequences.

Probes may also be used for the detection of related sequences, and mayhave at least 50% sequence identity to any of the GCREC encodingsequences. The hybridization probes of the subject invention may be DNAor RNA and may be derived from the sequence of SEQ ID NO:11-20 or fromgenomic sequences including promoters, enhancers, and introns of theGCREC gene.

Means for producing specific hybridization probes for DNAs encodingGCREC include the cloning of polynucleotide sequences encoding GCREC orGCREC derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art, are commercially available, and may beused to synthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³²P or ³⁵S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

Polynucleotide sequences encoding GCREC may be used for the diagnosis ofdisorders associated with expression of GCREC. Examples of suchdisorders include, but are not limited to, a cell proliferative disordersuch as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis,cirrhosis, hepatitis, mixed connective tissue disease (MCID),myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera,psoriasis, primary thrombocythemia, and cancers includingadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; a neurological disordersuch as epilepsy, ischemic cerebrovascular disease, stroke, cerebralneoplasms, Alzheimer's disease, Pick's disease, Huntington's disease,dementia, Parkinson's disease and other extrapyramidal disorders,amyotrophic lateral sclerosis and other motor neuron disorders,progressive neural muscular atrophy, retinitis pigmentosa, hereditaryataxias, multiple sclerosis and other demyelinating diseases, bacterialand viral meningitis, brain abscess, subdural empyema, epidural abscess,suppurative intracranial thrombophlebitis, myelitis and radiculitis,viral central nervous system disease, prion diseases including kuru,Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome,fatal familial insomnia, nutritional and metabolic diseases of thenervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinalhemangioblastomatosis, encephalotrigeminal syndrome, mental retardationand other developmental disorders of the central nervous system,cerebral palsy, neuroskeletal disorders, autonomic nervous systemdisorders, cranial nerve disorders, spinal cord diseases, musculardystrophy and other neuromuscular disorders, peripheral nervous systemdisorders, dermatomyositis and polymyositis, inherited, metabolic,endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis,mental disorders including mood, anxiety, and schizophrenic disorders,seasonal affective disorder (SAD), akathesia, amnesia, catatonia,diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses,postherpetic neuralgia, Tourette's disorder, progressive supranuclearpalsy, corticobasal degeneration, and familial frontotemporal dementia;a cardiovascular disorder such as arteriovenous fistula,atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms,arterial dissections, varicose veins, thrombophlebitis andphlebothrombosis, vascular tumors, complications of thrombolysis,balloon angioplasty, vascular replacement, and coronary artery bypassgraft surgery, congestive heart failure, ischemic heart disease, anginapectoris, myocardial infarction, hypertensive heart disease,degenerative valvular heart disease, calcific aortic valve stenosis,congenitally bicuspid aortic valve, mitral annular calcification, mitralvalve prolapse, rheumatic fever and rheumatic heart disease, infectiveendocarditis, nonbacterial thrombotic endocarditis, endocarditis ofsystemic lupus erythematosus, carcinoid heart disease, cardiomyopathy,myocarditis, pericarditis, neoplastic heart disease, congenital heartdisease, and complications of cardiac transplantation; agastrointestinal disorder such as dysphagia, peptic esophagitis,esophageal spasm, esophageal stricture, esophageal carcinoma, dyspepsia,indigestion, gastritis, gastric carcinoma, anorexia, nausea, emesis,gastroparesis, antral or pyloric edema, abdominal angina, pyrosis,gastroenteritis, intestinal obstruction, infections of the intestinaltract, peptic ulcer, cholelithiasis, cholecystitis, cholestasis,pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis,hyperbilirubinemia, cirrhosis, passive congestion of the liver,hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis,Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, coloniccarcinoma, colonic obstruction, irritable bowel syndrome, short bowelsyndrome, diarrhea, constipation, gastrointestinal hemorrhage, acquiredimmunodeficiency syndrome (AIDS) enteropathy, jaundice, hepaticencephalopathy, hepatorenal syndrome, hepatic steatosis,hemochromatosis, Wilson's disease, alpha₁-antitrypsin deficiency, Reye'ssyndrome, primary sclerosing cholangitis, liver infarction, portal veinobstruction and thrombosis, centrilobular necrosis, peliosis hepatis,hepatic vein thrombosis, veno-occlusive disease, preeclampsia,eclampsia, acute fatty liver of pregnancy, intrahepatic cholestasis ofpregnancy, and hepatic tumors including nodular hyperplasias, adenomas,and carcinomas; an autoimmune/inflammatory disorder such as acquiredimmunodeficiency syndrome (AIDS), Addison's disease, adult respiratorydistress syndrome, allergies, ankylosing spondylitis, amyloidosis,anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmunethyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermaldystrophy (APECED), bronchitis, cholecystitis, contact dermatitis,Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosisfetalis, erythema nodosum, atrophic gastritis, glomerulonephritis,Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis,hypereosinophilia, irritable bowel syndrome, multiple sclerosis,myasthenia gravis, myocardial or pericardial inflammation,osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis,Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjögren'ssyndrome, systemic anaphylaxis, systemic lupus erythematosus, systemicsclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Wernersyndrome, complications of cancer, hemodialysis, and extracorporealcirculation, viral, bacterial, fungal, parasitic, protozoal, andhelminthic infections, and trauma; a metabolic disorder such asdiabetes, obesity, and osteoporosis; and an infection by a viral agentclassified as adenovirus, arenavirus, bunyavirus, calicivirus,coronavirus, filovirus, hepadnavirus, herpesvirus, flavivirus,orthomyxovirus, parvovirus, papovavirus, paramyxovirus, picomavirus,poxvirus, reovirus, retrovirus, rhabdovirus, and tongavirus. Thepolynucleotide sequences encoding GCREC may be used in Southern ornorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; in dipstick, pin, and multiformat ELISA-like assays;and in microarrays utilizing fluids or tissues from patients to detectaltered GCREC expression. Such qualitative or quantitative methods arewell known in the art.

In a particular aspect, the nucleotide sequences encoding GCREC may beuseful in assays that detect the presence of associated disorders,particularly those mentioned above. The nucleotide sequences encodingGCREC may be labeled by standard methods and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantified and compared with a standardvalue. If the amount of signal in the patient sample is significantlyaltered in comparison to a control sample then the presence of alteredlevels of nucleotide sequences encoding GCREC in the sample indicatesthe presence of the associated disorder. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or to monitor the treatment of anindividual patient.

In order to provide a basis for the diagnosis of a disorder associatedwith expression of GCREC, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, encoding GCREC, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withvalues from an experiment in which a known amount of a substantiallypurified polynucleotide is used. Standard values obtained in this mannermay be compared with values obtained from samples from patients who aresymptomatic for a disorder. Deviation from standard values is used toestablish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis todetermine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to cancer, the presence of an abnormal amount of transcript(either under- or overexpressed) in biopsied tissue from an individualmay indicate a predisposition for the development of the disease, or mayprovide a means for detecting the disease prior to the appearance ofactual clinical symptoms. A more definitive diagnosis of this type mayallow health professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding GCREC may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding GCREC, or a fragment of a polynucleotide complementary to thepolynucleotide encoding GCREC, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantification of closely related DNA or RNA sequences.

In a particular aspect, oligonucleotide primers derived from thepolynucleotide sequences encoding GCREC may be used to detect singlenucleotide polymorphisms (SNPs). SNPs are substitutions, insertions anddeletions that are a frequent cause of inherited or acquired geneticdisease in humans. Methods of SNP detection include, but are not limitedto, single-stranded conformation polymorphism (SSCP) and fluorescentSSCP (fSSCP) methods. In SSCP, oligonucleotide primers derived from thepolynucleotide sequences encoding GCREC are used to amplify DNA usingthe polymerase chain reaction (PCR). The DNA may be derived, forexample, from diseased or normal tissue, biopsy samples, bodily fluids,and the like. SNPs in the DNA cause differences in the secondary andtertiary structures of PCR products in single-stranded form, and thesedifferences are detectable using gel electrophoresis in non-denaturinggels. In fSCCP, the oligonucleotide primers are fluorescently labeled,which allows detection of the amplimers in high-throughput equipmentsuch as DNA sequencing machines. Additionally, sequence databaseanalysis methods, termed in silico SNP (isSNP), are capable ofidentifying polymorphisms by comparing the sequence of individualoverlapping DNA fragments which assemble into a common consensussequence. These computer-based methods filter out sequence variationsdue to laboratory preparation of DNA and sequencing errors usingstatistical models and automated analyses of DNA sequence chromatograms.In the alternative, SNPs may be detected and characterized by massspectrometry using, for example, the high throughput MASSARRAY system(Sequenom, Inc., San Diego Calif.).

Methods which may also be used to quantify the expression of GCRECinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and interpolating results from standard curves.(See, e.g., Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244;Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236.) The speed ofquantitation of multiple samples may be accelerated by running the assayin a high-throughput format where the oligomer or polynucleotide ofinterest is presented in various dilutions and a spectrophotometric orcalorimetric response gives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotide sequences described herein may be used aselements on a nicroarray. The rnicroarray can be used in transcriptimaging techniques which monitor the relative expression levels of largenumbers of genes simultaneously as described below. The microarray mayalso be used to identify genetic variants, mutations, and polymorphisms.This information may be used to determine gene function, to understandthe genetic basis of a disorder, to diagnose a disorder, to monitorprogression/regression of disease as a function of gene expression, andto develop and monitor the activities of therapeutic agents in thetreatment of disease. In particular, this information may be used todevelop a pharmacogenomic profile of a patient in order to select themost appropriate and effective treatment regimen for that patient. Forexample, therapeutic agents which are highly effective and display thefewest side effects may be selected for a patient based on his/herpharmacogenomic profile.

In another embodiment, GCREC, fragments of GCREC, or antibodies specificfor GCREC may be used as elements on a microarray. The microarray may beused to monitor or measure protein-protein interactions, drug-targetinteractions, and gene expression profiles, as described above.

A particular embodiment relates to the use of the polynucleotides of thepresent invention to generate a transcript image of a tissue or celltype. A transcript image represents the global pattern of geneexpression by a particular tissue or cell type. Global gene expressionpatterns are analyzed by quantifying the number of expressed genes andtheir relative abundance under given conditions and at a given time.(See Seilhamer et al., “Comparative Gene Transcript Analysis,” U.S. Pat.No. 5,840,484, expressly incorporated by reference herein.) Thus atranscript image may be generated by hybridizing the polynucleotides ofthe present invention or their complements to the totality oftranscripts or reverse transcripts of a particular tissue or cell type.In one embodiment, the hybridization takes place in high-throughputformat, wherein the polynucleotides of the present invention or theircomplements comprise a subset of a plurality of elements on amicroarray. The resultant transcript image would provide a profile ofgene activity.

Transcript images may be generated using transcripts isolated fromtissues, cell lines, biopsies, or other biological samples. Thetranscript image may thus reflect gene expression in vivo as in the caseof a tissue or biopsy sample, or in vitro, as in the case of a cellline.

Transcript images which profile the expression of the polynucleotides ofthe present invention may also be used in conjunction with in vitromodel systems and preclinical evaluation of pharmaceuticals, as well astoxicological testing of industrial and naturally-occurringenvironmental compounds. All compounds induce characteristic geneexpression patterns, frequently termed molecular fingerprints ortoxicant signatures, which are indicative of mechanisms of action andtoxicity (Nuwaysir, E. F. et al. (1999) Mol. Carcinog. 24:153-159;Steiner, S. and N. L. Anderson (2000) Toxicol. Lett. 112-113:467-471,expressly incorporated by reference herein). If a test compound has asignature similar to that of a compound with known toxicity, it islikely to share those toxic properties. These fingerprints or signaturesare most useful and refined when they contain expression informationfrom a large number of genes and gene families. Ideally, a genome-widemeasurement of expression provides the highest quality signature. Evengenes whose expression is not altered by any tested compounds areimportant as well, as the levels of expression of these genes are usedto normalize the rest of the expression data. The normalizationprocedure is useful for comparison of expression data after treatmentwith different compounds. While the assignment of gene function toelements of a toxicant signature aids in interpretation of toxicitymechanisms, knowledge of gene function is not necessary for thestatistical matching of signatures which leads to prediction oftoxicity. (See, for example, Press Release 00-02 from the NationalInstitute of Environmental Health Sciences, released Feb. 29, 2000,available at http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore,it is important and desirable in toxicological screening using toxicantsignatures to include all expressed gene sequences.

In one embodiment, the toxicity of a test compound is assessed bytreating a biological sample containing nucleic acids with the testcompound. Nucleic acids that are expressed in the treated biologicalsample are hybridized with one or more probes specific to thepolynucleotides of the present invention, so that transcript levelscorresponding to the polynucleotides of the present invention may bequantified. The transcript levels in the treated biological sample arecompared with levels in an untreated biological sample. Differences inthe transcript levels between the two samples are indicative of a toxicresponse caused by the test compound in the treated sample.

Another particular embodiment relates to the use of the polypeptidesequences of the present invention to analyze the proteome of a tissueor cell type. The term proteome refers to the global pattern of proteinexpression in a particular tissue or cell type. Each protein componentof a proteome can be subjected individually to further analysis.Proteome expression patterns, or profiles, are analyzed by quantifyingthe number of expressed proteins and their relative abundance undergiven conditions and at a given time. A profile of a cell's proteome maythus be generated by separating and analyzing the polypeptides of aparticular tissue or cell type. In one embodiment, the separation isachieved using two-dimensional gel electrophoresis, in which proteinsfrom a sample are separated by isoelectric focusing in the firstdimension, and then according to molecular weight by sodium dodecylsulfate slab gel electrophoresis in the second dimension (Steiner andAnderson, supra). The proteins are visualized in the gel as discrete anduniquely positioned spots, typically by staining the gel with an agentsuch as Coomassie Blue or silver or fluorescent stains. The opticaldensity of each protein spot is generally proportional to the level ofthe protein in the sample. The optical densities of equivalentlypositioned protein spots from different samples, for example, frombiological samples either treated or untreated with a test compound ortherapeutic agent, are compared to identify any changes in protein spotdensity related to the treatment. The proteins in the spots arepartially sequenced using, for example, standard methods employingchemical or enzymatic cleavage followed by mass spectrometry. Theidentity of the protein in a spot may be determined by comparing itspartial sequence, preferably of at least 5 contiguous amino acidresidues, to the polypeptide sequences of the present invention. In somecases, further sequence data may be obtained for definitive proteinidentification.

A proteomic profile may also be generated using antibodies specific forGCREC to quantify the levels of GCREC expression. In one embodiment, theantibodies are used as elements on a microarray, and protein expressionlevels are quantified by exposing the microarray to the sample anddetecting the levels of protein bound to each array element (Lueking, A.et al. (1999) Anal. Biochem. 270:103-111; Mendoze, L. G. et al. (1999)Biotechniques 27:778-788). Detection may be performed by a variety ofmethods known in the art, for example, by reacting the proteins in thesample with a thiol- or amino-reactive fluorescent compound anddetecting the amount of fluorescence bound at each array element.

Toxicant signatures at the proteome level are also useful fortoxicological screening, and should be analyzed in parallel withtoxicant signatures at the transcript level. There is a poor correlationbetween transcript and protein abundances for some proteins in sometissues (Anderson, N. L. and J. Seilhamer (1997) Electrophoresis18:533-537), so proteome toxicant signatures may be useful in theanalysis of compounds which do not significantly affect the transcriptimage, but which alter the proteomic profile. In addition, the analysisof transcripts in body fluids is difficult, due to rapid degradation ofmRNA, so proteomic profiling may be more reliable and informative insuch cases.

In another embodiment, the toxicity of a test compound is assessed bytreating a biological sample containing proteins with the test compound.Proteins that are expressed in the treated biological sample areseparated so that the amount of each protein can be quantified. Theamount of each protein is compared to the amount of the correspondingprotein in an untreated biological sample. A difference in the amount ofprotein between the two samples is indicative of a toxic response to thetest compound in the treated sample. Individual proteins are identifiedby sequencing the amino acid residues of the individual proteins andcomparing these partial sequences to the polypeptides of the presentinvention.

In another embodiment, the toxicity of a test compound is assessed bytreating a biological sample containing proteins with the test compound.Proteins from the biological sample are incubated with antibodiesspecific to the polypeptides of the present invention. The amount ofprotein recognized by the antibodies is quantified. The amount ofprotein in the treated biological sample is compared with the amount inan untreated biological sample. A difference in the amount of proteinbetween the two samples is indicative of a toxic response to the testcompound in the treated sample.

Microarrays may be prepared, used, and analyzed using methods known inthe art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No.5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA93:10614-10619; Baldeschweiler et al. (1995) PCT applicationWO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; andHeller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.) Various types ofmicroarrays are well known and thoroughly described in DNA Microarrays:A Practical Approach, M. Schena, ed. (1999) Oxford University Press,London, hereby expressly incorporated by reference.

In another embodiment of the invention, nucleic acid sequences encodingGCREC may be used to generate hybridization probes useful in mapping thenaturally occurring genomic sequence. Either coding or noncodingsequences may be used, and in some instances, noncoding sequences may bepreferable over coding sequences. For example, conservation of a codingsequence among members of a multi-gene family may potentially causeundesired cross hybridization during chromosomal mapping. The sequencesmay be mapped to a particular chromosome, to a specific region of achromosome, or to artificial chromosome constructions, e.g., humanartificial chromosomes (HACs), yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs), bacterial P1 constructions, orsingle chromosome cDNA libraries. (See, e.g., Harrington, J. J. et al.(1997) Nat. Genet. 15:345-355; Price, C. M. (1993) Blood Rev. 7:127-134;and Trask, B. J. (1991) Trends Genet. 7:149-154.) Once mapped, thenucleic acid sequences of the invention may be used to develop geneticlinkage maps, for example, which correlate the inheritance of a diseasestate with the inheritance of a particular chromosome region orrestriction fragment length polymorphism (RFLP). (See, for example,Lander, E. S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA83:7353-7357.)

Fluorescent in situ hybridization (FISH) may be correlated with otherphysical and genetic map data. (See, e.g., Heinz-Ulrich, et al. (1995)in Meyers, supra, pp. 965-968.) Examples of genetic map data can befound in various scientific journals or at the Online MendelianInheritance in Man (OMIM) World Wide Web site. Correlation between thelocation of the gene encoding GCREC on a physical map and a specificdisorder, or a predisposition to a specific disorder, may help definethe region of DNA associated with that disorder and thus may furtherpositional cloning efforts.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the exact chromosomal locus is notknown. This information is valuable to investigators searching fordisease genes using positional cloning or other gene discoverytechniques. Once the gene or genes responsible for a disease or syndromehave been crudely localized by genetic linkage to a particular genomicregion, e.g., ataxia-telangiectasia to 11q22-23, any sequences mappingto that area may represent associated or regulatory genes for furtherinvestigation. (See, e.g., Gatti, R. A. et al. (1988) Nature336:577-580.) The nucleotide sequence of the instant invention may alsobe used to detect differences in the chromosomal location due totranslocation, inversion, etc., among normal, carrier, or affectedindividuals.

In another embodiment of the invention, GCREC, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes betweenGCREC and the agent being tested may be measured.

Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geysen, et al. (1984) PCT applicationWO84/03564.) In this method, large numbers of different small testcompounds are synthesized on a solid substrate. The test compounds arereacted with GCREC, or fragments thereof, and washed. Bound GCREC isthen detected by methods well known in the art. Purified GCREC can alsobe coated directly onto plates for use in the aforementioned drugscreening techniques. Alternatively, non-neutralizing antibodies can beused to capture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding GCREC specificallycompete with a test compound for binding GCREC. In this manner,antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with GCREC.

In additional embodiments, the nucleotide sequences which encode GCRECmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following embodiments are, therefore, to beconstrued as merely illustrative, and not limitative of the remainder ofthe disclosure in any way whatsoever.

The disclosures of all patents, applications and publications, mentionedabove and below, including U.S. Ser. No. 60/212,483, U.S. Ser. No.60/213,950, U.S. Ser. No. 60/214,062, U.S. Ser. No. 60/216,595, U.S.Ser. No. 60/218,936, and U.S. Ser. No. 60/219,154, are expresslyincorporated by reference herein.

EXAMPLES

I. Construction of cDNA Libraries

Incyte cDNAs were derived from cDNA libraries described in the LIFESEQGOLD database (Incyte Genomics, Palo Alto Calif.) and shown in Table 4,column 5. Some tissues were homogenized and lysed in guanidiniumisothiocyanate, while others were homogenized and lysed in phenol or ina suitable mixture of denaturants, such as TRIZOL (Life Technologies), amonophasic solution of phenol and guanidine isothiocyanate. Theresulting lysates were centrifuged over CsCl cushions or extracted withchloroform. RNA was precipitated from the lysates with eitherisopropanol or sodium acetate and ethanol, or by other routine methods.

Phenol extraction and precipitation of RNA were repeated as necessary toincrease RNA purity. In some cases, RNA was treated with DNase. For mostlibraries, poly(A)+RNA was isolated using oligo d(T)-coupledparamagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN,Chatsworth Calif.), or an OLIGOTEX mRNA purification kit (QIAGEN).Alternatively, RNA was isolated directly from tissue lysates using otherRNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion,Austin Tex.).

In some cases, Stratagene was provided with RNA and constructed thecorresponding cDNA libraries. Otherwise, cDNA was synthesized and cDNAlibraries were constructed with the UNIAP vector system (Stratagene) orSUPERSCRIPT plasmid system (Life Technologies), using the recommendedprocedures or similar methods known in the art. (See, e.g., Ausubel,1997, supra, units 5.1-6.6.) Reverse transcription was initiated usingoligo d(T) or random primers. Synthetic oligonucleotide adapters wereligated to double stranded cDNA, and the cDNA was digested with theappropriate restriction enzyme or enzymes. For most libraries, the cDNAwas size-selected (300-1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B,or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) orpreparative agarose gel electrophoresis. cDNAs were ligated intocompatible restriction enzyme sites of the polylinker of a suitableplasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (LifeTechnologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad Calif.), PBK-CMVplasmid (Stratagene), or pINCY (Incyte Genomics, Palo Alto Calif.), orderivatives thereof. Recombinant plasmids were transformed intocompetent E. coli cells including XL1-Blue, XL1-BlueMRF, or SOLR fromStratagene or DH5α, DH10B, or ElectroMAX DH10B from Life Technologies.

II. Isolation of cDNA Clones

Plasmids obtained as described in Example I were recovered from hostcells by in vivo excision using the UNIZAP vector system (Stratagene) orby cell lysis. Plasmids were purified using at least one of thefollowing: a Magic or WIZARD Minipreps DNA purification system(Promega); an AGTC Miniprep purification kit (Edge Biosystems,Gaithersburg Md.); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid,QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96plasmid purification kit from QIAGEN. Following precipitation, plasmidswere resuspended in 0.1 ml of distilled water and stored, with orwithout lyophilization, at 4° C.

Alternatively, plasmid DNA was amplified from host cell lysates usingdirect link PCR in a high-throughput format (Rao, V. B. (1994) Anal.Biochem. 216:1-14). Host cell lysis and thermal cycling steps werecarried out in a single reaction mixture. Samples were processed andstored in 384-well plates, and the concentration of amplified plasmidDNA was quantified fluorometrically using PICOGREEN dye (MolecularProbes, Eugene Oreg.) and a FLUOROSKAN II fluorescence scanner(Labsystems Oy, Helsinki, Finland).

III. Sequencing and Analysis

Incyte cDNA recovered in plasmids as described in Example II weresequenced as follows. Sequencing reactions were processed using standardmethods or high-throughput instrumentation such as the ABI CATALYST 800(Applied Biosystems) thermal cycler or the PTC-200 thermal cycler (MJResearch) in conjunction with the HYDRA microdispenser (RobbinsScientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNAsequencing reactions were prepared using reagents provided by AmershamPharmacia Biotech or supplied in ABI sequencing kits such as the ABIPRISM BIGDYE Terminator cycle sequencing ready reaction kit (AppliedBiosystems). Electrophoretic separation of cDNA sequencing reactions anddetection of labeled polynucleotides were carried out using the MEGABACE1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or377 sequencing system (Applied Biosystems) in conjunction with standardABI protocols and base calling software; or other sequence analysissystems known in the art. Reading frames within the cDNA sequences wereidentified using standard methods (reviewed in Ausubel, 1997, supra,unit 7.7). Some of the cDNA sequences were selected for extension usingthe techniques disclosed in Example VIII.

The polynucleotide sequences derived from Incyte cDNAs were validated byremoving vector, linker, and poly(A) sequences and by masking ambiguousbases, using algorithms and programs based on BLAST, dynamicprogramming, and dinucleotide nearest neighbor analysis. The Incyte cDNAsequences or translations thereof were then queried against a selectionof public databases such as the GenBank primate, rodent, mammalian,vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM,and hidden Markov model (HMM)-based protein family databases such asPFAM. (HMM is a probabilistic approach which analyzes consensus primarystructures of gene families. See, for example, Eddy, S. R. (1996) Curr.Opin. Struct. Biol. 6:361-365.) The queries were performed usingprograms based on BLAST, FASTA, BLIMPS, and HMMER. The Incyte cDNAsequences were assembled to produce full length polynucleotidesequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitchedsequences, stretched sequences, or Genscan-predicted coding sequences(see Examples IV and V) were used to extend Incyte cDNA assemblages tofull length. Assembly was performed using programs based on Phred,Phrap, and Consed, and cDNA assemblages were screened for open readingframes using programs based on GeneMark, BLAST, and FASTA. The fulllength polynucleotide sequences were translated to derive thecorresponding full length polypeptide sequences. Alternatively, apolypeptide of the invention may begin at any of the methionine residuesof the full length translated polypeptide. Full length polypeptidesequences were subsequently analyzed by querying against databases suchas the GenBank protein databases (genpept), SwissProt, BLOCKS, PRINTS,DOMO, PRODOM, Prosite, and hidden Markov model (IHM)-based proteinfamily databases such as PFAM. Full length polynucleotide sequences arealso analyzed using MACDNASIS PRO software (Hitachi SoftwareEngineering, South San Francisco Calif.) and LASERGENE software(DNASTAR). Polynucleotide and polypeptide sequence alignments aregenerated using default parameters specified by the CLUSTAL algorithm asincorporated into the MEGALIGN multisequence alignment program(DNASTAR), which also calculates the percent identity between alignedsequences.

Table 7 summarizes the tools, programs, and algorithms used for theanalysis and assembly of Incyte cDNA and full length sequences andprovides applicable descriptions, references, and threshold parameters.The first column of Table 7 shows the tools, programs, and algorithmsused, the second column provides brief descriptions thereof, the thirdcolumn presents appropriate references, all of which are incorporated byreference herein in their entirety, and the fourth column presents,where applicable, the scores, probability values, and other parametersused to evaluate the strength of a match between two sequences (thehigher the score or the lower the probability value, the greater theidentity between two sequences).

The programs described above for the assembly and analysis of fulllength polynucleotide and polypeptide sequences were also used toidentify polynucleotide sequence fragments from SEQ ID NO:11-20.Fragments from about 20 to about 4000 nucleotides which are useful inhybridization and amplification technologies are described in Table 4,column 4.

IV. Identification and Editing of Coding Sequences from Genomic DNA

Putative G-protein coupled receptors were initially identified byrunning the Genscan gene identification program against public genomicsequence databases (e.g., gbpri and gbhtg). Genscan is a general-purposegene identification program which analyzes genomic DNA sequences from avariety of organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol.268:78-94, and Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol.8:346-354). The program concatenates predicted exons to form anassembled cDNA sequence extending from a methionine to a stop codon. Theoutput of Genscan is a FASTA database of polynucleotide and polypeptidesequences. The maximum range of sequence for Genscan to analyze at oncewas set to 30 kb. To determine which of these Genscan predicted cDNAsequences encode G-protein coupled receptors, the encoded polypeptideswere analyzed by querying against PFAM models for G-protein coupledreceptors. Potential G-protein coupled receptors were also identified byhomology to Incyte cDNA sequences that had been annotated as G-proteincoupled receptors. These selected Genscan-predicted sequences were thencompared by BLAST analysis to the genpept and gbpri public databases.Where necessary, the Genscan-predicted sequences were then edited bycomparison to the top BLAST hit from genpept to correct errors in thesequence predicted by Genscan, such as extra or omitted exons. BLASTanalysis was also used to find any Incyte cDNA or public cDNA coverageof the Genscan-predicted sequences, thus providing evidence fortranscription. When Incyte cDNA coverage was available, this informationwas used to correct or confirm the Genscan predicted sequence. Fulllength polynucleotide sequences were obtained by assemblingGenscan-predicted coding sequences with Incyte cDNA sequences and/orpublic cDNA sequences using the assembly process described in ExampleIII. Alternatively, full length polynucleotide sequences were derivedentirely from edited or unedited Genscan-predicted coding sequences.

V. Assembly of Genomic Sequence Data with cDNA Sequence Data “Stitched”Sequences

Partial cDNA sequences were extended with exons predicted by the Genscangene identification program described in Example IV. Partial cDNAsassembled as described in Example III were mapped to genomic DNA andparsed into clusters containing related cDNAs and Genscan exonpredictions from one or more genomic sequences. Each cluster wasanalyzed using an algorithm based on graph theory and dynamicprogramming to integrate cDNA and genomic information, generatingpossible splice variants that were subsequently confirmed, edited, orextended to create a full length sequence. Sequence intervals in whichthe entire length of the interval was present on more than one sequencein the cluster were identified, and intervals thus identified wereconsidered to be equivalent by transitivity. For example, if an intervalwas present on a cDNA and two genomic sequences, then all threeintervals were considered to be equivalent. This process allowsunrelated but consecutive genomic sequences to be brought together,bridged by cDNA sequence. Intervals thus identified were then “stitched”together by the stitching algorithm in the order that they appear alongtheir parent sequences to generate the longest possible sequence, aswell as sequence variants. Linkages between intervals which proceedalong one type of parent sequence (cDNA to cDNA or genomic sequence togenomic sequence) were given preference over linkages which changeparent type (cDNA to genomic sequence). The resultant stitched sequenceswere translated and compared by BLAST analysis to the genpept and gbpripublic databases. Incorrect exons predicted by Genscan were corrected bycomparison to the top BLAST hit from genpept. Sequences were furtherextended with additional cDNA sequences, or by inspection of genomicDNA, when necessary.

“Stretched” Sequences

Partial DNA sequences were extended to full length with an algorithmbased on BLAST analysis. First, partial cDNAs assembled as described inExample III were queried against public databases such as the GenBankprimate, rodent, mammalian, vertebrate, and eukaryote databases usingthe BLAST program. The nearest GenBank protein homolog was then comparedby BLAST analysis to either Incyte cDNA sequences or GenScan exonpredicted sequences described in Example IV. A chimeric protein wasgenerated by using the resultant high-scoring segment pairs (HSPs) tomap the translated sequences onto the GenBank protein homolog.Insertions or deletions may occur in the chimeric protein with respectto the original GenBank protein homolog. The GenBank protein homolog,the chimeric protein, or both were used as probes to search forhomologous genomic sequences from the public human genome databases.Partial DNA sequences were therefore “stretched” or extended by theaddition of homologous genomic sequences. The resultant stretchedsequences were examined to determine whether it contained a completegene.

VI. Chromosomal Mapping of GCREC Encoding Polynucleotides

The sequences which were used to assemble SEQ ID NO:11-20 were comparedwith sequences from the Incyte LIFESEQ database and public domaindatabases using BLAST and other implementations of the Smith-Watermanalgorithm. Sequences from these databases that matched SEQ ID NO:11-20were assembled into clusters of contiguous and overlapping sequencesusing assembly algorithms such as Phrap (Table 7). Radiation hybrid andgenetic mapping data available from public resources such as theStanford Human Genome Center (SHGC), Whitehead Institute for GenomeResearch (WIGR), and Genethon were used to determine if any of theclustered sequences had been previously mapped. Inclusion of a mappedsequence in a cluster resulted in the assignment of all sequences ofthat cluster, including its particular SEQ ID NO:, to that map location.

Map locations are represented by ranges, or intervals, of humanchromosomes. The map position of an interval, in centiMorgans, ismeasured relative to the terminus of the chromosome's p-arm. (ThecentiMorgan (cM) is a unit of measurement based on recombinationfrequencies between chromosomal markers. On average, 1 cM is roughlyequivalent to 1 megabase (Mb) of DNA in humans, although this can varywidely due to hot and cold spots of recombination.) The cM distances arebased on genetic markers mapped by Genethon which provide boundaries forradiation hybrid markers whose sequences were included in each of theclusters. Human genome maps and other resources available to the public,such as the NCBI “GeneMap'99” World Wide Web site(http://www.ncbi.nlm.nih.gov/genemap/), can be employed to determine ifpreviously identified disease genes map within or in proximity to theintervals indicated above.

VII. Analysis of Polynucleotide Expression

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7;Ausubel (1995) supra, ch. 4 and 16.)

Analogous computer techniques applying BLAST were used to search foridentical or related molecules in cDNA databases such as GenBank orLIFESEQ (Incyte Genomics). This analysis is much faster than multiplemembrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or similar. The basis of the search is theproduct score, which is defined as:

$\frac{{BLAST}\mspace{14mu} {Score} \times {Percent}\mspace{14mu} {Identity}}{5 \times {minimum}\left\{ {{{length}\left( {{Seq}{.1}} \right)},{{length}\left( {{Seq}{.2}} \right)}} \right\}}$

The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. The productscore is a normalized value between 0 and 100, and is calculated asfollows: the BLAST score is multiplied by the percent nucleotideidentity and the product is divided by (5 times the length of theshorter of the two sequences). The BLAST score is calculated byassigning a score of +5 for every base that matches in a high-scoringsegment pair (HSP), and −4 for every mismatch. Two sequences may sharemore than one HSP (separated by gaps). If there is more than one HSP,then the pair with the highest BLAST score is used to calculate theproduct score. The product score represents a balance between fractionaloverlap and quality in a BLAST alignment. For example, a product scoreof 100 is produced only for 100% identity over the entire length of theshorter of the two sequences being compared. A product score of 70 isproduced either by 100% identity and 70% overlap at one end, or by 88%identity and 100% overlap at the other. A product score of 50 isproduced either by 100% identity and 50% overlap at one end, or 79%identity and 100% overlap.

Alternatively, polynucleotide sequences encoding GCREC are analyzed withrespect to the tissue sources from which they were derived. For example,some full length sequences are assembled, at least in part, withoverlapping Incyte cDNA sequences (see Example III). Each cDNA sequenceis derived from a cDNA library constructed from a human tissue. Eachhuman tissue is classified into one of the following organ/tissuecategories: cardiovascular system; connective tissue; digestive system;embryonic structures; endocrine system; exocrine glands; genitalia,female; genitalia, male; germ cells; hemic and immune system; liver;musculoskeletal system; nervous system; pancreas; respiratory system;sense organs; skin; stomatognathic system; unclassified/mixed; orurinary tract. The number of libraries in each category is counted anddivided by the total number of libraries across all categories.Similarly, each human tissue is classified into one of the followingdisease/condition categories: cancer, cell line, developmental,inflammation, neurological, trauma, cardiovascular, pooled, and other,and the number of libraries in each category is counted and divided bythe total number of libraries across all categories. The resultingpercentages reflect the tissue- and disease-specific expression of cDNAencoding GCREC. cDNA sequences and cDNA library/tissue information arefound in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto Calif.).

VIII. Extension of GCREC Encoding Polynucleotides

Full length polynucleotide sequences were also produced by extension ofan appropriate fragment of the full length molecule usingoligonucleotide primers designed from this fragment. One primer wassynthesized to initiate 5′ extension of the known fragment, and theother primer was synthesized to initiate 3′ extension of the knownfragment. The initial primers were designed using OLIGO 4.06 software(National Biosciences), or another appropriate program, to be about 22to 30 nucleotides in length, to have a GC content of about 50% or more,and to anneal to the target sequence at temperatures of about 68° C. toabout 72° C. Any stretch of nucleotides which would result in hairpinstructures and primer-primer dimerizations was avoided.

Selected human cDNA libraries were used to extend the sequence. If morethan one extension was necessary or desired, additional or nested setsof primers were designed.

High fidelity amplification was obtained by PCR using methods well knownin the art. PCR was performed in 96-well plates using the PTC-200thermal cycler (MJ Research, Inc.). The reaction mix contained DNAtemplate, 200 nmol of each primer, reaction buffer containing Mg²⁺,(NH₄)₂SO₄, and 2-mercaptoethanol, Taq DNA polymerase (Amersham PharmaciaBiotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase(Stratagene), with the following parameters for primer pair PCI A andPCI B: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 60° C., 1min; Step 4: 68° C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times;Step 6: 68° C., 5 min; Step 7: storage at 4° C. In the alternative, theparameters for primer pair T7 and SK+ were as follows: Step 1: 94° C., 3min; Step 2: 94° C., 15 sec; Step 3: 57° C., 1 min; Step 4: 68° C., 2min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68° C., 5 min;Step 7: storage at 4° C.

The concentration of DNA in each well was determined by dispensing 100μl PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; MolecularProbes, Eugene Oreg.) dissolved in 1TE and 0.5 μl of undiluted PCRproduct into each well of an opaque fluorimeter plate (Corning Costar,Acton Mass.), allowing the DNA to bind to the reagent. The plate wasscanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measurethe fluorescence of the sample and to quantify the concentration of DNA.A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a 1% agarose gel to determine which reactions weresuccessful in extending the sequence.

The extended nucleotides were desalted and concentrated, transferred to384-well plates, digested with CviJI cholera virus endonuclease(Molecular Biology Research, Madison Wis.), and sonicated or shearedprior to religation into pUC 18 vector (Amersham Pharmacia Biotech). Forshotgun sequencing, the digested nucleotides were separated on lowconcentration (0.6 to 0.8%) agarose gels, fragments were excised, andagar digested with Agar ACE (Promega). Extended clones were religatedusing T4 ligase (New England Biolabs, Beverly Mass.) into pUC 18 vector(Amersham Pharmacia Biotech), treated with Pfu DNA polymerase(Stratagene) to fill-in restriction site overhangs, and transfected intocompetent E. coli cells. Transformed cells were selected onantibiotic-containing media, and individual colonies were picked andcultured overnight at 37° C. in 384-well plates in LB/2x carb liquidmedia.

The cells were lysed, and DNA was amplified by PCR using Taq DNApolymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase(Stratagene) with the following parameters: Step 1: 94° C., 3 min; Step2: 94° C., 15 sec; Step 3: 60° C., 1 min; Step 4: 72° C., 2 min; Step 5:steps 2,3, and 4 repeated 29 times; Step 6: 72° C., 5 min; Step 7:storage at 4° C. DNA was quantified by PICOGREEN reagent (MolecularProbes) as described above. Samples with low DNA recoveries werereamplified using the same conditions as described above. Samples werediluted with 20% dimethysulfoxide (1:2, v/v), and sequenced usingDYENAMIC energy transfer sequencing primers and the DYNAMIC DIRECT kit(Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cyclesequencing ready reaction kit (Applied Biosystems).

In like manner, full length polynucleotide sequences are verified usingthe above procedure or are used to obtain 5′ regulatory sequences usingthe above procedure along with oligonucleotides designed for suchextension, and an appropriate genomic library.

IX. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:11-20 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer, 250 μCi of [γ-³²P] adenosinetriphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase(DuPont NEN, Boston Mass.). The labeled oligonucleotides aresubstantially purified using a SEPHADEX G-25 superfine size exclusiondextran bead column (Amersham Pharmacia Biotech). An aliquot containing10⁷ counts per minute of the labeled probe is used in a typicalmembrane-based hybridization analysis of human genomic DNA digested withone of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba I,or Pvu II (DuPont NEN).

The DNA from each digest is fractionated on a 0.7% agarose gel andtransferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under conditions of up to, for example, 0.1× saline sodiumcitrate and 0.5% sodium dodecyl sulfate. Hybridization patterns arevisualized using autoradiography or an alternative imaging means andcompared.

X. Microarrays

The linkage or synthesis of array elements upon a microarray can beachieved utilizing photolithography, piezoelectric printing (ink-jetprinting, See, e.g., Baldeschweiler, supra.), mechanical microspottingtechnologies, and derivatives thereof. The substrate in each of theaforementioned technologies should be uniform and solid with anon-porous surface (Schena (1999), supra). Suggested substrates includesilicon, silica, glass slides, glass chips, and silicon wafers.Alternatively, a procedure analogous to a dot or slot blot may also beused to arrange and link elements to the surface of a substrate usingthermal, UV, chemical, or mechanical bonding procedures. A typical arraymay be produced using available methods and machines well known to thoseof ordinary skill in the art and may contain any appropriate number ofelements. (See, e.g., Schena, M. et al. (1995) Science 270:467-470;Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J.Hodgson (1998) Nat. Biotechnol. 16:27-31.)

Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments oroligomers thereof may comprise the elements of the microarray. Fragmentsor oligomers suitable for hybridization can be selected using softwarewell known in the art such as LASERGENE software (DNASTAR). The arrayelements are hybridized with polynucleotides in a biological sample. Thepolynucleotides in the biological sample are conjugated to a fluorescentlabel or other molecular tag for ease of detection. After hybridization,nonhybridized nucleotides from the biological sample are removed, and afluorescence scanner is used to detect hybridization at each arrayelement. Alternatively, laser desorbtion and mass spectrometry may beused for detection of hybridization. The degree of complementarity andthe relative abundance of each polynucleotide which hybridizes to anelement on the microarray may be assessed. In one embodiment, microarraypreparation and usage is described in detail below.

Tissue or Cell Sample Preparation

Total RNA is isolated from tissue samples using the guanidiniumthiocyanate method and poly(A)⁺ RNA is purified using the oligo-(dT)cellulose method. Each poly(A)⁺ RNA sample is reverse transcribed usingMMLV reverse-transcriptase, 0.05 pg/μl oligo-(dT) primer (21mer), 1×first strand buffer, 0.03 units/μl RNase inhibitor, 500 μM DATP, 500 μMdGTP, 500 μM dTIP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5(Amersham Pharmacia Biotech). The reverse transcription reaction isperformed in a 25 ml volume containing 200 ng poly(A)⁺ RNA withGEMBRIGHT kits (Incyte). Specific control poly(A)⁺ RNAs are synthesizedby in vitro transcription from non-coding yeast genomic DNA. Afterincubation at 37° C. for 2 hr, each reaction sample (one with Cy3 andanother with Cy5 labeling) is treated with 2.5 ml of 0.5M sodiumhydroxide and incubated for 20 minutes at 85° C. to the stop thereaction and degrade the RNA. Samples are purified using two successiveCHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc.(CLONTECH), Palo Alto Calif.) and after combining, both reaction samplesare ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodiumacetate, and 300 ml of 100% ethanol. The sample is then dried tocompletion using a SpeedVAC (Savant Instruments Inc., Holbrook N.Y.) andresuspended in 14 μl 5×SSC/0.2% SDS.

Microarray Preparation

Sequences of the present invention are used to generate array elements.Each array element is amplified from bacterial cells containing vectorswith cloned cDNA inserts. PCR amplification uses primers complementaryto the vector sequences flanking the cDNA insert. Array elements areamplified in thirty cycles of PCR from an initial quantity of 1-2 ng toa final quantity greater than 5 μg. Amplified array elements are thenpurified using SEPHACRYL-400 (Amersham Pharmacia Biotech).

Purified array elements are immobilized on polymer-coated glass slides.Glass microscope slides (Corning) are cleaned by ultrasound in 0.1% SDSand acetone, with extensive distilled water washes between and aftertreatments. Glass slides are etched in 4% hydrofluoric acid (VWRScientific Products Corporation (VWR), West Chester Pa.), washedextensively in distilled water, and coated with 0.05% aminopropyl silane(Sigma) in 95% ethanol. Coated slides are cured in a 110° C. oven.

Array elements are applied to the coated glass substrate using aprocedure described in U.S. Pat. No. 5,807,522, incorporated herein byreference. 1 μl of the array element DNA, at an average concentration of100 ng/μl, is loaded into the open capillary printing element by ahigh-speed robotic apparatus. The apparatus then deposits about 5 nl ofarray element sample per slide.

Microarrays are UV-crosslinked using a STRATALNKER UV-crosslinker(Stratagene). Microarrays are washed at room temperature once in 0.2%SDS and three times in distilled water. Non-specific binding sites areblocked by incubation of microarrays in 0.2% casein in phosphatebuffered saline (PBS) (Tropix, Inc., Bedford Mass.) for 30 minutes at60° C. followed by washes in 0.2% SDS and distilled water as before.

Hybridization

Hybridization reactions contain 9 μl of sample mixture consisting of 0.2μg each of Cy3 and Cy5 labeled cDNA synthesis products in 5×SSC, 0.2%SDS hybridization buffer. The sample mixture is heated to 65° C. for 5minutes and is aliquoted onto the microarray surface and covered with an1.8 cm² coverslip. The arrays are transferred to a waterproof chamberhaving a cavity just slightly larger than a microscope slide. Thechamber is kept at 100% humidity internally by the addition of 140 μl of5×SSC in a corner of the chamber. The chamber containing the arrays isincubated for about 6.5 hours at 60° C. The arrays are washed for 10 minat 45° C. in a first wash buffer (1×SSC, 0.1% SDS), three times for 10minutes each at 45° C. in a second wash buffer (0.1×SSC), and dried.

Detection

Reporter-labeled hybridization complexes are detected with a microscopeequipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., SantaClara Calif.) capable of generating spectral lines at 488 nm forexcitation of Cy3 and at 632 nm for excitation of Cy5. The excitationlaser light is focused on the array using a 20× microscope objective(Nikon, Inc., Melville N.Y.). The slide containing the array is placedon a computer-controlled X-Y stage on the microscope and raster-scannedpast the objective. The 1.8 cm×1.8 cm array used in the present exampleis scanned with a resolution of 20 micrometers.

In two separate scans, a mixed gas multiline laser excites the twofluorophores sequentially. Emitted light is split, based on wavelength,into two photomultiplier tube detectors (PMT R1477, Hamamatsu PhotonicsSystems, Bridgewater N.J.) corresponding to the two fluorophores.Appropriate filters positioned between the array and the photomultipliertubes are used to filter the signals. The emission maxima of thefluorophores used are 565 nm for Cy3 and 650 nm for Cy5. Each array istypically scanned twice, one scan per fluorophore using the appropriatefilters at the laser source, although the apparatus is capable ofrecording the spectra from both fluorophores simultaneously.

The sensitivity of the scans is typically calibrated using the signalintensity generated by a cDNA control species added to the samplemixture at a known concentration. A specific location on the arraycontains a complementary DNA sequence, allowing the intensity of thesignal at that location to be correlated with a weight ratio ofhybridizing species of 1:100,000. When two samples from differentsources (e.g., representing test and control cells), each labeled with adifferent fluorophore, are hybridized to a single array for the purposeof identifying genes that are differentially expressed, the calibrationis done by labeling samples of the calibrating cDNA with the twofluorophores and adding identical amounts of each to the hybridizationmixture.

The output of the photomultiplier tube is digitized using a 12-bitRTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc.,Norwood Mass.) installed in an IBM-compatible PC computer. The digitizeddata are displayed as an image where the signal intensity is mappedusing a linear 20-color transformation to a pseudocolor scale rangingfrom blue (low signal) to red (high signal). The data is also analyzedquantitatively. Where two different fluorophores are excited andmeasured simultaneously, the data are first corrected for opticalcrosstalk (due to overlapping emission spectra) between the fluorophoresusing each fluorophore's emission spectrum.

A grid is superimposed over the fluorescence signal image such that thesignal from each spot is centered in each element of the grid. Thefluorescence signal within each element is then integrated to obtain anumerical value corresponding to the average intensity of the signal.The software used for signal analysis is the GEMTOOLS gene expressionanalysis program (Incyte).

XI. Complementary Polynucleotides

Sequences complementary to the GCREC-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring GCREC. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO 4.06 software(National Biosciences) and the coding sequence of GCREC. To inhibittranscription, a complementary oligonucleotide is designed from the mostunique 5′ sequence and used to prevent promoter binding to the codingsequence. To inhibit translation, a complementary oligonucleotide isdesigned to prevent ribosomal binding to the GCREC-encoding transcript.

XII. Expression of GCREC

Expression and purification of GCREC is achieved using bacterial orvirus-based expression systems. For expression of GCREC in bacteria,cDNA is subcloned into an appropriate vector containing an antibioticresistance gene and an inducible promoter that directs high levels ofcDNA transcription. Examples of such promoters include, but are notlimited to, the trp-lac (tac) hybrid promoter and the T5 or T7bacteriophage promoter in conjunction with the lac operator regulatoryelement. Recombinant vectors are transformed into suitable bacterialhosts, e.g., BL21(DE3). Antibiotic resistant bacteria express GCREC uponinduction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expressionof GCREC in eukaryotic cells is achieved by infecting insect ormammalian cell lines with recombinant Autographica californica nuclearpolyhedrosis virus (AcMNPV), commonly known as baculovirus. Thenonessential polyhedrin gene of baculovirus is replaced with cDNAencoding GCREC by either homologous recombination or bacterial-mediatedtransposition involving transfer plasmid intermediates. Viralinfectivity is maintained and the strong polyhedrin promoter drives highlevels of cDNA transcription. Recombinant baculovirus is used to infectSpodoptera frugiperda (Sf9) insect cells in most cases, or humanhepatocytes, in some cases. Infection of the latter requires additionalgenetic modifications to baculovirus. (See Engelhard, E. K. et al.(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)Hum. Gene Ther. 7:1937-1945.)

In most expression systems, GCREC is synthesized as a fusion proteinwith, e.g., glutathione S-transferase (GST) or a peptide epitope tag,such as FLAG or 6-His, permitting rapid, single-step, affinity-basedpurification of recombinant fusion protein from crude cell lysates. GST,a 26-kilodalton enzyme from Schistosoma japonicum, enables thepurification of fusion proteins on immobilized glutathione underconditions that maintain protein activity and antigenicity (AmershamPharmacia Biotech). Following purification, the GST moiety can beproteolytically cleaved from GCREC at specifically engineered sites.FLAG, an 8-amino acid peptide, enables immunoaffinity purification usingcommercially available monoclonal and polyclonal anti-FLAG antibodies(Eastman Kodak). 6-His, a stretch of six consecutive histidine residues,enables purification on metal-chelate resins (QIAGEN). Methods forprotein expression and purification are discussed in Ausubel (1995,supra, ch. 10 and 16). Purified GCREC obtained by these methods can beused directly in the assays shown in Examples XVI, XVII, and XVIII,where applicable.

XIII. Functional Assays

GCREC function is assessed by expressing the sequences encoding GCREC atphysiologically elevated levels in mammalian cell culture systems. cDNAis subcloned into a mammalian expression vector containing a strongpromoter that drives high levels of cDNA expression. Vectors of choiceinclude PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, CarlsbadCalif.), both of which contain the cytomegalovirus promoter. 5-10 μg ofrecombinant vector are transiently transfected into a human cell line,for example, an endothelial or hematopoietic cell line, using eitherliposome formulations or electroporation. 1-2 μg of an additionalplasmid containing sequences encoding a marker protein areco-transfected. Expression of a marker protein provides a means todistinguish transfected cells from nontransfected cells and is areliable predictor of cDNA expression from the recombinant vector.Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP;Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), anautomated, laser optics-based technique, is used to identify transfectedcells expressing GFP or CD64-GFP and to evaluate the apoptotic state ofthe cells and other cellular properties. FCM detects and quantifies theuptake of fluorescent molecules that diagnose events preceding orcoincident with cell death. These events include changes in nuclear DNAcontent as measured by staining of DNA with propidium iodide; changes incell size and granularity as measured by forward light scatter and 90degree side light scatter; down-regulation of DNA synthesis as measuredby decrease in bromodeoxyuridine uptake; alterations in expression ofcell surface and intracellular proteins as measured by reactivity withspecific antibodies; and alterations in plasma membrane composition asmeasured by the binding of fluorescein-conjugated Annexin V protein tothe cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York N.Y.

The influence of GCREC on gene expression can be assessed using highlypurified populations of cells transfected with sequences encoding GCRECand either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on thesurface of transfected cells and bind to conserved regions of humanimmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using magnetic beads coated with either human IgGor antibody against CD64 (DYNAL, Lake Success N.Y.). mRNA can bepurified from the cells using methods well known by those of skill inthe art. Expression of mRNA encoding GCREC and other genes of interestcan be analyzed by northern analysis or microarray techniques.

XIV. Production of GCREC Specific Antibodies

GCREC substantially purified using polyacrylamide gel electrophoresis(PAGE; see, e.g., Harrington, M. G. (1990) Methods Enzymol.182:488-495), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols.

Alternatively, the GCREC amino acid sequence is analyzed using LASERGENEsoftware (DNASTAR) to determine regions of high immunogenicity, and acorresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skill in the art. Methods for selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions are well described in the art. (See, e.g., Ausubel,1995, supra, ch. 11.)

Typically, oligopeptides of about 15 residues in length are synthesizedusing an ABI 431A peptide synthesizer (Applied Biosystems) using FMOCchemistry and coupled to KLH (Sigma-Aldrich, St. Louis Mo.) by reactionwith N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increaseimmunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunizedwith the oligopeptide-KLH complex in complete Freund's adjuvant.Resulting antisera are tested for antipeptide and anti-GCREC activityby, for example, binding the peptide or GCREC to a substrate, blockingwith 1% BSA, reacting with rabbit antisera, washing, and reacting withradio-iodinated goat anti-rabbit IgG.

XV. Purification of Naturally Occurring GCREC Using Specific Antibodies

Naturally occurring or recombinant GCREC is substantially purified byimmunoaffinity chromatography using antibodies specific for GCREC. Animmunoaffinity column is constructed by covalently coupling anti-GCRECantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin isblocked and washed according to the manufacturer's instructions.

Media containing GCREC are passed over the immunoaffinity column, andthe column is washed under conditions that allow the preferentialabsorbance of GCREC (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/GCREC binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), andGCREC is collected.

XVI. Identification of Molecules which Interact with GCREC

Molecules which interact with GCREC may include agonists andantagonists, as well as molecules involved in signal transduction, suchas G proteins. GCREC, or a fragment thereof, is labeled with ¹²⁵IBolton-Hunter reagent. (See, e.g., Bolton A. E. and W. M. Hunter (1973)Biochem. J. 133:529-539.) A fragment of GCREC includes, for example, afragment comprising one or more of the three extracellular loops, theextracellular N-terminal region, or the third intracellular loop.Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled GCREC, washed, and any wells withlabeled GCREC complex are assayed. Data obtained using differentconcentrations of GCREC are used to calculate values for the number,affinity, and association of GCREC with the candidate ligand molecules.

Alternatively, molecules interacting with GCREC are analyzed using theyeast two-hybrid system as described in Fields, S. and O. Song (1989)Nature 340:245-246, or using commercially available kits based on thetwo-hybrid system, such as the MATCHMAKER system (Clontech). GCREC mayalso be used in the PATHCALLING process (CuraGen Corp., New Haven Conn.)which employs the yeast two-hybrid system in a high-throughput manner todetermine all interactions between the proteins encoded by two largelibraries of genes (Nandabalan, K. et al. (2000) U.S. Pat. No.6,057,101).

Potential GCREC agonists or antagonists may be tested for activation orinhibition of GCREC receptor activity using the assays described insections XVII and XVIII. Candidate molecules may be selected from knownGPCR agonists or antagonists, peptide libraries, or combinatorialchemical libraries.

Methods for detecting interactions of GCREC with intracellular signaltransduction molecules such as G proteins are based on the premise thatinternal segments or cytoplasmic domains from an orphan Gprotein-coupled seven transmembrane receptor may be exchanged with theanalogous domains of a known G protein-coupled seven transmembranereceptor and used to identify the G-proteins and downstream signalingpathways activated by the orphan receptor domains (Kobilka, B. K. et al.(1988) Science 240:1310-1316). In an analogous fashion, domains of theorphan receptor may be cloned as a portion of a fusion protein and usedin binding assays to demonstrate interactions with specific G proteins.Studies have shown that the third intracellular loop of Gprotein-coupled seven transmembrane receptors is important for G proteininteraction and signal transduction (Conklin, B. R. et al. (1993) Cell73:631-641). For example, the DNA fragment corresponding to the thirdintracellular loop of GCREC may be amplified by the polymerase chainreaction (PCR) and subcloned into a fusion vector such as pGEX(Pharmacia Biotech). The construct is transformed into an appropriatebacterial host, induced, and the fusion protein is purified from thecell lysate by glutathione-Sepharose 4B (Pharmacia Biotech) affinitychromatography.

For in vitro binding assays, cell extracts containing G proteins areprepared by extraction with 50 mM Tris, pH 7.8, 1 mM EGTA, 5 mM MgCl₂,20 mM CHAPS, 20% glycerol, 10 μg of both aprotinin and leupeptin, and 20μl of 50 mM phenylmethylsulfonyl fluoride. The lysate is incubated onice for 45 min with constant stirring, centrifuged at 23,000 g for 15min at 4° C., and the supernatant is collected. 750 μg of cell extractis incubated with glutathione S-transferase (GST) fusion protein beadsfor 2 h at 4° C. The GST beads are washed five times withphosphate-buffered saline. Bound G subunits are detected by[³²P]ADP-ribosylation with pertussis or cholera toxins. The reactionsare terminated by the addition of SDS sample buffer (4.6% (w/v) SDS, 10%(v/v) β-mercaptoethanol, 20% (w/v) glycerol, 95.2 mM Tris-HCl, pH 6.8,0.01% (w/v) bromphenol blue). The [³²P]ADP-labeled proteins areseparated on 10% SDS-PAGE gels, and autoradiographed. The separatedproteins in these gels are transferred to nitrocellulose paper, blockedwith blotto (5% nonfat dried milk, 50 mM Tris-HCl (pH 8.0), 2 mM CaCl₂,80 mM NaCl, 0.02% NaN₃, and 0.2% Nonidet P-40) for 1 hour at roomtemperature, followed by incubation for 1.5 hours with G a subtypeselective antibodies (1:500; Calbiochem-Novabiochem). After threewashes, blots are incubated with horseradish peroxidase (HIW)-conjugatedgoat anti-rabbit immunoglobulin (1:2000, Cappel, Westchester Pa.) andvisualized by the chemiluminescence-based ECL method (Amersham Corp.).

XVII. Demonstration of GCREC Activity

An assay for GCREC activity measures the expression of GCREC on the cellsurface. cDNA encoding GCREC is transfected into an appropriatemammalian cell line. Cell surface proteins are labeled with biotin asdescribed (de la Fuente, M. A. et al. (1997) Blood 90:2398-2405).Immunoprecipitations are performed using GCREC-specific antibodies, andimmunoprecipitated samples are analyzed using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and immunoblottingtechniques. The ratio of labeled immunoprecipitant to unlabeledimmunoprecipitant is proportional to the amount of GCREC expressed onthe cell surface.

In the alternative, an assay for GCREC activity is based on aprototypical assay for ligand/receptor-mediated modulation of cellproliferation. This assay measures the rate of DNA synthesis in Swissmouse 3T3 cells. A plasmid containing polynucleotides encoding GCREC isadded to quiescent 3T3 cultured cells using transfection methods wellknown in the art. The transiently transfected cells are then incubatedin the presence of [³H]thymidine, a radioactive DNA precursor molecule.Varying amounts of GCREC ligand are then added to the cultured cells.Incorporation of [³H]thymidine into acid-precipitable DNA is measuredover an appropriate time interval using a radioisotope counter, and theamount incorporated is directly proportional to the amount of newlysynthesized DNA. A linear dose-response curve over at least ahundred-fold GCREC ligand concentration range is indicative of receptoractivity. One unit of activity per milliliter is defined as theconcentration of GCREC producing a 50% response level, where 100%represents maximal incorporation of [³H]thymidine into acid-precipitableDNA (McKay, I. and I. Leigh, eds. (1993) Growth Factors: A PracticalApproach, Oxford University Press, New York N.Y., p. 73.)

In a further alternative, the assay for GCREC activity is based upon theability of GPCR family proteins to modulate G protein-activated secondmessenger signal transduction pathways (e.g., cAMP; Gaudin, P. et al.(1998) J. Biol. Chem. 273:4990-4996). A plasmid encoding full lengthGCREC is transfected into a mammalian cell line (e.g., Chinese hamsterovary (CHO) or human embryonic kidney (HEK-293) cell lines) usingmethods well-known in the art. Transfected cells are grown in 12-welltrays in culture medium for 48 hours, then the culture medium isdiscarded, and the attached cells are gently washed with PBS. The cellsare then incubated in culture medium with or without ligand for 30minutes, then the medium is removed and cells lysed by treatment with 1M perchloric acid. The cAMP levels in the lysate are measured byradioimmunoassay using methods well-known in the art. Changes in thelevels of cAMP in the lysate from cells exposed to ligand compared tothose without ligand are proportional to the amount of GCREC present inthe transfected cells.

To measure changes in inositol phosphate levels, the cells are grown in24-well plates containing 1×10⁵ cells/well and incubated withinositol-free media and [³H]myoinositol, 2 μCi/well, for 48 hr. Theculture medium is removed, and the cells washed with buffer containing10 mM LiCl followed by addition of ligand. The reaction is stopped byaddition of perchloric acid. Inositol phosphates are extracted andseparated on Dowex AGI-X8 (Bio-Rad) anion exchange resin, and the totallabeled inositol phosphates counted by liquid scintillation. Changes inthe levels of labeled inositol phosphate from cells exposed to ligandcompared to those without ligand are proportional to the amount of GCRECpresent in the transfected cells.

XVII. Identification of GCREC Ligands

GCREC is expressed in a eukaryotic cell line such as CHO (ChineseHamster Ovary) or HEK (Human Embryonic Kidney) 293 which have a goodhistory of GPCR expression and which contain a wide range of G-proteinsallowing for functional coupling of the expressed GCREC to downstreameffectors. The transformed cells are assayed for activation of theexpressed receptors in the presence of candidate ligands. Activity ismeasured by changes in intracellular second messengers, such as cyclicAMP or Ca²⁺. These may be measured directly using standard methods wellknown in the art, or by the use of reporter gene assays in which aluminescent protein (e.g. firefly luciferase or green fluorescentprotein) is under the transcriptional control of a promoter responsiveto the stimulation of protein kinase C by the activated receptor(Milligan, G. et al. (1996) Trends Pharmacol. Sci. 17:235-237). Assaytechnologies are available for both of these second messenger systems toallow high throughput readout in multi-well plate format, such as theadenylyl cyclase activation FlashPlate Assay (NEN Life SciencesProducts), or fluorescent Ca²⁺ indicators such as Fluo-4 AM (MolecularProbes) in combination with the FLIPR fluorimetric plate reading system(Molecular Devices). In cases where the physiologically relevant secondmessenger pathway is not known, GCREC may be coexpressed with theG-proteins G_(α15/16) which have been demonstrated to couple to a widerange of G-proteins (Offermanns, S. and M. I. Simon (1995) J. Biol.Chem. 270:15175-15180), in order to funnel the signal transduction ofthe GCREC through a pathway involving phospholipase C and Ca²⁺mobilization. Alternatively, GCREC may be expressed in engineered yeastsystems which lack endogenous GPCRs, thus providing the advantage of anull background for GCREC activation screening. These yeast systemssubstitute a human GPCR and G. protein for the corresponding componentsof the endogenous yeast pheromone receptor pathway. Downstream signalingpathways are also modified so that the normal yeast response to thesignal is converted to positive growth on selective media or to reportergene expression (Broach, J. R. and J. Thomer (1996) Nature 384 (supp.):14-16). The receptors are screened against putative ligands includingknown GPCR ligands and other naturally occurring bioactive molecules.Biological extracts from tissues, biological fluids and cellsupernatants are also screened.

Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with certain embodiments,it should be understood that the invention as claimed should not beunduly limited to such specific embodiments. Indeed, variousmodifications of the described modes for carrying out the inventionwhich are obvious to those skilled in molecular biology or relatedfields are intended to be within the scope of the following claims.

TABLE 1 Incyte Incyte Incyte Polypeptide Polypeptide PolynucleotidePolynucleotide Project ID SEQ ID NO: ID SEQ ID NO: ID 7474927 17474927CD1 11 7474927CB1 7475194 2 7475194CD1 12 7475194CB1 7475203 37475203CD1 13 7475203CB1 7474987 4 7474987CD1 14 7474987CB1 5617631 55617631CD1 15 5617631CB1 7472098 6 7472098CD1 16 7472098CB1 7476775 77476775CD1 17 7476775CB1 7477937 8 7477937CD1 18 7477937CB1 7476798 97476798CD1 19 7476798CB1 7477889 10 7477889CD1 20 7477889CB1

TABLE 2 Incyte Polypeptide Polypeptide GenBank Probability GenBank SEQID NO: ID ID NO: Score Homolog 1 7474927CD1 g3892596 6.20E−26 [Musmusculus] pheromone receptor 2 (seven domain GPCR) g10732802 0 [Homosapiens] vomeronasal receptor 1 2 7475194CD1 g683747 2.10E−100 [Homosapiens] extracellular calcium-sensing receptor (GPCR) (Garrett, J. E.et al. (1995) J. Biol. Chem. 270: 12919-12925) g13936377 0 [Musmusculus] taste receptor T1R3 3 7475203CD1 g12745520 0 [Mus musculus]putative sweet taste receptor T1R1 g1836094 1.70E−110 [Homo sapiens]calcium-sensing receptor, CaSR, human, medullary (GPCR) (Freichel, M. etal. (1996) Endocrinology 137: 3842-3848) 4 7474987CD1 g6691938 7.20E−85[Homo sapiens] novel 7 transmembrane receptor g7638409 4.60E−67 [Musmusculus] olfactory receptor P2 (Zheng, C. et al. (2000) Neuron 26:81-91) 5 5617631CD1 g7638409 2.20E−67 [Mus musculus] olfactory receptorP2 (Zheng, C. et al. (2000) Neuron 26: 81-91) g12007428 8.00E−73 [Musmusculus] B5 olfactory receptor 6 7472098CD1 g11908221 1.00E−89 [Musmusculus] MOR 3′Beta6 g4761598 3.70E−81 [Mus musculus] MOR 3′Beta2(Bulger, M. et al. (1999) Proc. Natl. Acad. Sci. USA 96: 5129-5134) 77476775CD1 g4680254 1.10E−145 [Mus musculus] odorant receptor S1 87477937CD1 g2765660 5.90E−76 [Gallus gallus] chick olfactory receptor 7(Nef, S. and P. Nef. (1997) Proc. Natl. Acad. Sci. USA 94: 4766-4771) 97476798CD1 g12007423 2.00E−81 [Mus musculus] T2 olfactory receptorg7638409 8.90E−73 [Mus musculus] olfactory receptor P2 10 7477889CD1g7638409 2.80E−62 [Mus musculus] olfactory receptor P2

TABLE 3 Incyte Potential Potential Analytical Polypeptide Amino AcidPhosphorylation Glycosylation Signature Sequences, Methods and SEQ IDNO: ID Residues Sites Sites Domains and Motifs Databases 1 7474927CD1353 T5, S164, S200, N117, N183, RECEPTOR PHEROMONE, G-PROTEIN BLAST-S209, S213, N198, N256 COUPLED VN1 VN2 VN3 VN7 VN4 VN5: PRODOM S217,S258, PD009900: I73-F335 S262, T341 G-protein coupled receptor: BLIMPS-BL00237C: S266-L292 BLOCKS 2 7475194CD1 863 T102, T153, N85, N130,Metabotropic glutamate GPCR BLIMPS- S175, S189, N264, N285, signature:PR00248A: K32-G44; PRINTS S214, S289, N380, N411, PR00248B: G69-N84;PR00248C: N84-C103; S293, S477, N432, N475, PR00248D: V141-Y167; T480,S539, N748 PR00248E: L174-Q193; PR00248F: S562, S570, Q193-V209;PR00248G: V209-F226; S678, PR00248H: S607-C629; PR00248J: A692-P715;PR00248K: T747-N770; PR00248L: N770-P791; PR00592D: N130-G143; PR00592E:D215-C236 Transmembrane domains: L581-F601; HMMER L617-F635; A692-L711Receptor family ligand binding HMMER-PFAM region (ANF_receptor):V61-D470 G-protein coupled receptor: BLIMPS- BL00979A: L71-A118;BL00979B: S147-L194; BLOCKS BL00979C: T195-F226;; BL00979F: G384-K422;BL00979I: P506-H526; BL00979J: Y530-L581; BL00979K: L586-V632; BL00979L:L633-S673; BL00979M: A746-V796 RECEPTOR, G-PROTEIN COUPLED, BLAST-TRANSMEMBRANE GLYCOPROTEIN, PRODOM PHEROMONE PRECURSOR, METABOTROPICGLUTAMATE: PD001315: W575-N835 G-PROTEIN COUPLED RECEPTORS FAMILYBLAST-DOMO 3: DM00837|I59362|1-893: L9-H341 G-protein coupled receptormotif MOTIFS (0754.pdoc): C528-C552 3 7475203CD1 841 T5, S28, T60, N87,N88, G-protein coupled receptor: BLIMPS- S67, T115, N95, N291, BL00979A:H74-L121; BL00979B: T149-V196; BLOCKS T149, S177, N479, N529, BL00979C:E197-L228; S216, S275, N822 BL00979G: V428-D455; BL00979I: T293, Y341,P493-H513; BL00979K: A573-L619; T650, S663, BL00979L: Y620-F660;BL00979M: S781, S830, L733-Y783; BL00979N: Y787-S823 T835 Metabotropicglutamate GPCR BLIMPS- signature: PR00248A: P35-H47; PRINTS PR00248B:G72-N87; PR00248C: N87-C106; PR00248D: V143-Y169; PR00248E: L176-Q195;PR00248F: Q195-I211; PR00248G: I211-L228; PR00248H: T594-S616; PR00248I:A639-F660; PR00248J: A679-T702; PR00248K: Y734-N757; PR00248L: N757-T778Signal peptide: M1-S25 SIGPEPT Signal cleavage: M1-S25 SPSCANTransmembrane domains: V569-W590; HMMER L681-W701; T763-Y783 Receptorfamily ligand binding HMMER-PFAM region (ANF_receptor): C66-E480 7transmembrane receptor HMMER-PFAM (metabotropic (7tm_3)): A572-N822RECEPTOR, G-PROTEIN COUPLED; BLAST- TRANSMEMBRANE GLYCOPROTEIN; PRODOMPHEROMONE PRECURSOR; SIGNAL METABOTROPIC GLUTAMATE GPCR: PD001315:V569-N822 G-PROTEIN COUPLED RECEPTORS FAMILY BLAST-DOMO 3:DM00837|P35384|1-894: L363-S830 4 7474987CD1 309 T47 S65 S186 N3 N63 N87transmembrane domains: I27-I46; HMMER S265 T17 T222 N88 I195-I214 S288T307 7 transmembrane receptor (rhodopsin HMMER-PFAM family): G39-Y287G-protein coupled receptor: BLIMPS- BL00237A: N88-P127; BL00237C:S16-L42; BLOCKS BL00237D: P279-K295 G-protein coupled receptorsPROFILESCAN signature: F100-V145 G_Protein_Receptor: A108-I124 MOTIFSOlfactory receptor signature: BLIMPS- PR00245A: V57-K78; PR00245B:F175-E189; PRINTS PR00245C: Y236-T251; PR00245D: I271-F282; PR00245E:S288-I302 Rhodopsin-like GPCR superfamily BLIMPS- signature: PRINTSPR00237A: L24-T48; PR00237B: V57-K78; PR00237C: L102-I124; PR00237E:L197-F220; PR00237F: E194-H218; PR00237G: A269-K295 RECEPTOR OLFACTORYPROTEIN G- BLAST- PROTEIN COUPLED TRANSMEMBRANE PRODOM GLYCOPROTEINMULTIGENE FAMILY PD000921: L164-L244 G-PROTEIN COUPLED RECEPTORSBLAST-DOMO DM00013|P23274|18-306: L28-L298 DM00013|S29707|18-306:L28-L301 DM00013|P23266|17-306: I27-L298 DM00013|P23269|15-304: L28-L2985 5617631CD1 317 S22, S50, S68, N4 Olfactory receptor signature: BLIMPS-S138, S164, PR00245A: M60-Q81; PR00245B: F178-D192; PRINTS S189, S233,PR00245C: F239-T254; S292 PR00245D: F275-C286; PR00245E: S292-L306OLFACTORY RECEPTOR PROTEIN, BLAST- RECEPTOR-LIKE G-PROTEIN COUPLEDPRODOM TRANSMEMBRANE GLYCOPROTEIN, MULTI- GENE FAMILY: PD149621:T247-R308 G-protein Receptor: A111-I127 MOTIFS G-PROTEIN COUPLEDRECEPTORS: BLAST-DOMO DM00013|P23270|18-311: R24-L306 Signal cleavage:M1-A40 SPSCAN Transmembrane domain: A25-I48; M60-I79; HMMER F195-T215 7transmembrane receptor (rhodopsin HMMER-PFAM family), 7tm_1: M41-Y291G-protein coupled receptor: BLIMPS- BL00237A: H91-P130; BL00237D:T283-K299 BLOCKS G-protein coupled receptors PROFILESCAN signature:Y103-T148 6 7472098CD1 317 S180, T191 N44 G-PROTEIN COUPLED RECEPTORS:BLAST-DOMO DM00013|G45774|18-309: P20-L306 PUTATIVE G-PROTEIN COUPLEDBLAST- RECEPTOR, RA1C: PD170483: I251-I312 PRODOM Transmembrane domain:F207-F225, HMMER M36-R55 7 transmembrane receptor (rhodopsin HMMER-PFAMfamily), 7tm_1: G43-Y295 G-protein coupled receptor: BLIMPS- BL00237A:C92-P131; BL00237C: E235-S261; BLOCKS BL00237D: P287-R303 Olfactoryreceptor signature: BLIMPS- PR00245A: M61-K82; PR00245B: S180-D194;PRINTS PR00245C: L279-L290 7 7476775CD1 324 T147 T301 N12 Transmembranedomain: I35-G51 HMMER 7 transmembrane receptor (rhodopsin HMMER-PFAMfamily): G51-Y300 G-protein coupled receptors ProfileScan signature:Y112-F157 G-protein coupled receptor motif: MOTIFS T120-I136 G-proteincoupled receptor BLIMPS- signatures: BLOCKS BL00237A: K100-P139BL00237C: R245-S271 BL00237D: T292-K308 Olfactory receptor signatures:BLIMPS- PR00245A: M69-N90 PRINTS PR00245B: F187-P201 PR00245C: F248-G263PR00245D: L284-F295 PR00245E: T301-L315 G-protein coupled receptor:BLAST-DOMO DM00013|P23270|18-311: F27-L315 G-protein coupled receptor:BLAST-DOMO DM00013|P23267|20-309: F27-L315 G-protein coupled receptor:BLAST-DOMO DM00013|P23266|17-306: Q34-L315 G-protein coupled receptor:BLAST-DOMO DM00013|P30955|18-305: F38-L315 Olfactory G-protein coupledBLAST- receptor: PD000921: L176-V256 PRODOM Olfactory G-protein coupledBLAST- receptor: PD149621: V257-L315 PRODOM 8 7477937CD1 322 S164 S232S291 N5 G_Protein_Receptor: T110-I126 MOTIFS S316 S67 T237 G-proteincoupled receptors PROFILESCAN T315 signature: Y102-V147 Visual pigments(opsins) retinal PROFILESCAN binding site opsin.prf: S263-R318 signalpeptide: M136-T155 HMMER transmembrane domains: HMMER A25-T47, I92-M118,V197-I221 7 transmembrane receptor (rhodopsin HMMER-PFAM family) 7tm_1:G41-Y290 G-protein coupled receptor domain: BLIMPS- BL00237: Q90-P129,F200-F211, T195-I221, BLOCKS T282-K298 Olfactory receptor signatureBLIMPS- PR00245: M59-L80, F177-R191, F238-G253, PRINTS I274-L285,S291-M305 OLFACTORY RECEPTOR PROTEIN BLAST- PD000921: L166-L245, PRODOMPD149621: V247-K308 G-PROTEIN COUPLED RECEPTORS BLAST-DOMODM00013|P23274|18-306: E22-M305 9 7476798CD1 312 S136 S20 S263 N41 N5N88 7 transmembrane receptor (rhodopsin HMMER-PFAM S266 S290 S304family) 7tm_1: G40-Y289 S309 S66 T7 G-PROTEIN COUPLED RECEPTORSBLAST-DOMO DM00013|S29707|18-306: V17-L300 RECEPTOR OLFACTORYRECEPTORLIKE BLAST- GPROTEIN COUPLED TRANSMEMBRANE PRODOM GLYCOPROTEINMULTIGENE FAMILY PD000921: I165-L244 OLFACTORY RECEPTOR RECEPTORLIKEBLAST- GPROTEIN COUPLED TRANSMEMBRANE PRODOM GLYCOPROTEIN MULTIGENEFAMILY PD149621: T245-R306 G-protein coupled receptor BL00237: BLIMPS-K89-P128, T281-M297 BLOCKS Rhodopsin-like GPCR superfamily BLIMPS-PR00237: F25-F49, M58-K79, F103-I125, PRINTS M139-V160, V198-L221,A236-R260, K271-M297 Olfactory receptor signature BLIMPS- PR00245:M58-K79, F176-D190, Y237-A252, PRINTS L273-L284, S290-S304 G-proteincoupled receptors PROFILESCAN signature: F101-T145 transmem_domain:F25-F49, F199-G218 HMMER G_Protein_Receptor: A109-I125 MOTIFS 107477889CD1 319 S294 S71 N9 7 transmembrane receptor (rhodopsinHMMER-PFAM family) 7tm_1: G45-Y293 G-PROTEIN COUPLED RECEPTORSBLAST-DOMO DM00013|P23270|18-311: L29-H309 RECEPTOR OLFACTORYRECEPTORLIKE BLAST- GPROTEIN COUPLED TRANSMEMBRANE PRODOM GLYCOPROTEINMULTIGENE FAMILY PD000921: L170-L248 G-protein coupled receptor BL00237:BLIMPS- K94-P133, E235-M261, A285-K301 BLOCKS Rhodopsin-like GPCRsuperfamily BLIMPS- PR00237: L63-K84, F108-I130, V202-I225, PRINTSA240-R264, T275-K301 Olfactory receptor signature BLIMPS- PR00245:L63-K84, I181-D195, F241-G256, PRINTS S294-F308 G-protein coupledreceptors PROFILESCAN signature: Y106-G156 transmembrane domains:G28-I51, HMMER V211-M231 G_Protein_Receptor: T114-I130 MOTIFS

TABLE 4 Incyte Polynucleotide Polynucleotide Sequence Selected Sequence5′ 3′ SEQ ID NO: ID Length Fragments Fragments Position Position 117474927CB1 2245 755-1136, 5805033T6 (BONRFET03) 1704 2168 1-643,7673613H1 (FIBPFEC01) 1465 1986 2090-2116, 5805033F6 (BONRFET03) 17112245 1-1710 55012833H1 1 780 6939423H1 (FTUBTUR01) 1129 1492FL7474927_g2822142_g4995709 394 1455 12 7475194CB1 2729 1276-1513,7669623H1 (NOSEDIC02) 2123 2729 1-1175,FL7475194_g7523967_000013_g5809686 1 2592 1622-2182, 2454-2729 137475203CB1 2759 1672-1979, 55002220H2 1409 2048 1-608, 55002212H2 1 5532089-2197, 55002204H2 1319 1965 740-1464, GBI:g7669574_edit 53 25782738-2759 g5110689 2292 2759 55002204J2 2229 2753 14 7474987CB1 945555-639, GNN.g7283250_000011_008 1 945 918-945 15 5617631CB1 15111115-1154, 6036056F8 (PITUNOT06) 389 1119 1-663,FL5617631-g7157997_000060-g6691937 558 1511 1478-1511 71700159V1 1 52816 7472098CB1 954 1-106, FL7472098CB1_00001 1 954 496-656 17 7476775CB1975 1-51, GNN:g7838156_edit 1 975 573-975 18 7477937CB1 969 920-969GNN.g8568403_000027_002 1 969 19 7476798CB1 939 1-838,GNN.g8052176_000007_002 1 939 885-939 20 7477889CB1 960 1-24,GNN.g8570522_024.edit 1 960 594-655, 810-960

TABLE 5 Polynucleotide Incyte Representative SEQ ID NO: Project IDLibrary 11 7474927CB1 BONRFET03 12 7475194CB1 NOSEDIC02 15 5617631CB1THYMNOR02

TABLE 6 Library Vector Library Description BONRFET03 pINCY Library wasconstructed using RNA isolated from rib bone tissue removed from aCaucasian male fetus who died from Patau's syndrome (trisomy 13) at20-weeks' gestation. NOSEDIC02 PSPORT1 This large size fractionatedlibrary was constructed using RNA isolated from nasal polyp tissue.THYMNOR02 pINCY The library was constructed using RNA isolated fromthymus tissue removed from a 2-year- old Caucasian female during athymectomy and patch closure of left atrioventricular fistula. Pathologyindicated there was no gross abnormality of the thymus. The patientpresented with congenital heart abnormalities. Patient history includeddouble inlet left ventricle and a rudimentary right ventricle, pulmonaryhypertension, cyanosis, subaortic stenosis, seizures, and a fracture ofthe skull base. Family history included reflux neuropathy.

TABLE 7 Program Description Reference ABI FACTURA A program that removesvector sequences and Applied Biosystems, Foster City, CA. masksambiguous bases in nucleic acid sequences. ABI/PARACEL A Fast DataFinder useful in comparing and Applied Biosystems, Foster City, CA; FDFannotating amino acid or nucleic acid sequences. Paracel Inc., Pasadena,CA. ABI A program that assembles nucleic acid sequences. AppliedBiosystems, Foster City, CA. AutoAssembler BLAST A Basic Local AlignmentSearch Tool useful in Altschul, S. F. et al. (1990) J. Mol. Biol.sequence similarity search for amino acid and 215: 403-410; Altschul, S.F. et al. (1997) nucleic acid sequences. BLAST includes five NucleicAcids Res. 25: 3389-3402. functions: blastp, blastn, blastx, tblastn,and tblastx. FASTA A Pearson and Lipman algorithm that searches forPearson, W. R. and D. J. Lipman (1988) Proc. similarity between a querysequence and a group of Natl. Acad Sci. USA 85: 2444-2448; Pearson, W.R. sequences of the same type. FASTA comprises as (1990) MethodsEnzymol. 183: 63-98; least five functions: fasta, tfasta, fastx, tfastx,and and Smith, T. F. and M. S. Waterman (1981) ssearch. Adv. Appl. Math.2: 482-489. BLIMPS A BLocks IMProved Searcher that matches a Henikoff,S. and J. G. Henikoff (1991) Nucleic sequence against those in BLOCKS,PRINTS, Acids Res. 19: 6565-6572; Henikoff, J. G. and DOMO, PRODOM, andPFAM databases to search S. Henikoff (1996) Methods Enzymol. for genefamilies, sequence homology, and structural 266: 88-105; and Attwood, T.K. et al. (1997) J. fingerprint regions. Chem. Inf. Comput. Sci. 37:417-424. HMMER An algorithm for searching a query sequence againstKrogh. A. et al. (1994) J. Mol. Biol. hidden Markov model (HMM)-baseddatabases of 235: 1501-1531; Sonnhammer, E. L. L. et al. protein familyconsensus sequences, such as PFAM. (1988) Nucleic Acids Res. 26:320-322; Durbin, R. et al. (1998) Our World View, in a Nutshell,Cambridge Univ. Press, pp. 1-350. ProfileScan An algorithm that searchesfor structural and sequence Gribskov, M. et al. (1988) CABIOS 4: 61-66;motifs in protein sequences that match sequence patterns Gribskov, M. etal. (1989) Methods Enzymol. defined in Prosite. 183: 146-159; Bairoch,A. et al. (1997) Nucleic Acids Res. 25: 217-221. Phred A base-callingalgorithm that examines automated Ewing, B. et al. (1998) Genome Res.sequencer traces with high sensitivity and probability. 8: 175-185;Ewing, B. and P. Green (1998) Genome Res. 8: 186-194. Phrap A PhilsRevised Assembly Program including SWAT and Smith, T. F. and M. S.Waterman (1981) Adv. CrossMatch, programs based on efficientimplementation Appl. Math. 2: 482-489; Smith, T. F. and M. S. Watermanof the Smith-Waterman algorithm, useful in searching (1981) J. Mol.Biol. 147: 195-197; sequence homology and assembling DNA sequences. andGreen, P., University of Washington, Seattle, WA. Consed A graphicaltool for viewing and editing Phrap assemblies. Gordon, D. et al. (1998)Genome Res. 8: 195-202. SPScan A weight matrix analysis program thatscans protein Nielson, H. et al. (1997) Protein Engineering sequencesfor the presence of secretory signal peptides. 10: 1-6; Claverie, J. M.and S. Audic (1997) CABIOS 12: 431-439. TMAP A program that uses weightmatrices to delineate Persson, B. and P. Argos (1994) J. Mol. Biol.transmembrane segments on protein sequences and 237: 182-192; Persson,B. and P. Argos (1996) determine orientation. Protein Sci. 5: 363-371.TMHMMER A program that uses a hidden Markov model (HMM) to Sonnhammer,E. L. et al. (1998) Proc. Sixth Intl. delineate transmembrane segmentson protein sequences Conf. on Intelligent Systems for Mol. Biol., anddetermine orientation. Glasgow et al., eds., The Am. Assoc. forArtificial Intelligence Press, Menlo Park, CA, pp. 175-182. Motifs Aprogram that searches amino acid sequences for patterns Bairoch, A. etal. (1997) Nucleic Acids Res. 25: 217-221; that matched those defined inProsite. Wisconsin Package Program Manual, version 9, page M51-59,Genetics Computer Group, Madison, WL Program Parameter Threshold ABIFACTURA ABI/PARACEL FDF Mismatch < 50% ABI AutoAssembler BLAST ESTs:Probability value = 1.0E−8 or less Full Length sequences: Probabilityvalue = 1.0E−10 or less FASTA ESTs: fasta E value = 1.06E−6 AssembledESTs: fasta Identity = 95% or greater and Match length = 200 bases orgreater; fastx E value = 1.0E−8 or less Full Length sequences: fastxscore = 100 or greater BLIMPS Probability value = 1.0E−3 or less HMMERPFAM hits: Probability value = 1.0E−3 or less Signal peptide hits: Score= 0 or greater ProfileScan Normalized quality score ≧ GCG- specified“HIGH” value for that particular Prosite motif. Generally, score =1.4-2.1. Phred Phrap Score = 120 or greater; Match length = 56 orgreater Consed SPScan Score = 3.5 or greater TMAP TMHMMER Motifs

TABLE 8 Polynucleotide SEQ ID NO: Tissues 14 15 Breast, Fat, Skin + −Muscle, Bone, Synovium, − − Connective tissue Pancreas, Liver,Gallbladder − + Brain: Amygdala, Thalamus, Hippocampus, − − Entorhinalcortex, Archaecortex Brain: Striatum, Caudate nucleus, − − Putamen,Dentate nucleus, Globus pallidus, Substantia innominata, Ralphe magnusKidney, Fetal colon, Small intestine, − − Ileum, Esophagus Fetal heart,Aorta, Coronary artery − − Fetal lung, Adult lung − − Placenta,Prostate, Uterus − − Olfactory bulb − −

1. An isolated polypeptide selected from the group consisting of: a) apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-10, b) a polypeptide comprising a naturallyoccurring amino acid sequence at least 90% identical to an amino acidsequence selected from the group consisting of SEQ ID NO:1-10, c) abiologically active fragment of a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO:1-10, and d) animmunogenic fragment of a polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO:1-10.
 2. An isolatedpolypeptide of claim 1 selected from the group consisting of SEQ IDNO:1-10.
 3. An isolated polynucleotide encoding a polypeptide ofclaim
 1. 4. An isolated polynucleotide encoding a polypeptide of claim2.
 5. An isolated polynucleotide of claim 4 selected from the groupconsisting of SEQ ID NO:11-20.
 6. A recombinant polynucleotidecomprising a promoter sequence operably linked to a polynucleotide ofclaim
 3. 7. A cell transformed with a recombinant polynucleotide ofclaim
 6. 8. A transgenic organism comprising a recombinantpolynucleotide of claim
 6. 9. A method for producing a polypeptide ofclaim 1, the method comprising: a) culturing a cell under conditionssuitable for expression of the polypeptide, wherein said cell istransformed with a recombinant polynucleotide, and said recombinantpolynucleotide comprises a promoter sequence operably linked to apolynucleotide encoding the polypeptide of claim 1, and b) recoveringthe polypeptide so expressed.
 10. An isolated antibody whichspecifically binds to a polypeptide of claim
 1. 11. An isolatedpolynucleotide selected from the group consisting of: a) apolynucleotide comprising a polynucleotide sequence selected from thegroup consisting of SEQ D NO:11-20, b) a polynucleotide comprising anaturally occurring polynucleotide sequence at least 90% identical to apolynucleotide sequence selected from the group consisting of SEQ IDNO:11-20, c) a polynucleotide complementary to a polynucleotide of a),d) a polynucleotide complementary to a polynucleotide of b), and e) anRNA equivalent of a)-d).
 12. An isolated polynucleotide comprising atleast 60 contiguous nucleotides of a polynucleotide of claim
 11. 13. Amethod for detecting a target polynucleotide in a sample, said targetpolynucleotide having a sequence of a polynucleotide of claim 11, themethod comprising: a) hybridizing the sample with a probe comprising atleast 20 contiguous nucleotides comprising a sequence complementary tosaid target polynucleotide in the sample, and which probe specificallyhybridizes to said target polynucleotide, under conditions whereby ahybridization complex is formed between said probe and said targetpolynucleotide or fragments thereof, and b) detecting the presence orabsence of said hybridization complex, and, optionally, if present, theamount thereof.
 14. A method of claim 13, wherein the probe comprises atleast 60 contiguous nucleotides.
 15. A method for detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 11, the method comprising: a) amplifyingsaid target polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 16. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 17. Acomposition of claim 16, wherein the polypeptide has an amino acidsequence selected from the group consisting of SEQ ID NO:1-10.
 18. Amethod for treating a disease or condition associated with decreasedexpression of functional GCREC, comprising administering to a patient inneed of such treatment the composition of claim
 16. 19. A method forscreening a compound for effectiveness as an agonist of a polypeptide ofclaim 1, the method comprising: a) exposing a sample comprising apolypeptide of claim 1 to a compound, and b) detecting agonist activityin the sample.
 20. A composition comprising an agonist compoundidentified by a method of claim 19 and a pharmaceutically acceptableexcipient.
 21. A method for treating a disease or condition associatedwith decreased expression of functional GCREC, comprising administeringto a patient in need of such treatment a composition of claim
 20. 22. Amethod for screening a compound for effectiveness as an antagonist of apolypeptide of claim 1, the method comprising: a) exposing a samplecomprising a polypeptide of claim 1 to a compound, and b) detectingantagonist activity in the sample.
 23. A composition comprising anantagonist compound identified by a method of claim 22 and apharmaceutically acceptable excipient.
 24. A method for treating adisease or condition associated with overexpression of functional GCREC,comprising administering to a patient in need of such treatment acomposition of claim
 23. 25. A method of screening for a compound thatspecifically binds to the polypeptide of claim 1, said method comprisingthe steps of: a) combining the polypeptide of claim 1 with at least onetest compound under suitable conditions, and b) detecting binding of thepolypeptide of claim 1 to the test compound, thereby identifying acompound that specifically binds to the polypeptide of claim
 1. 26. Amethod of screening for a compound that modulates the activity of thepolypeptide of claim 1, said method comprising: a) combining thepolypeptide of claim 1 with at least one test compound under conditionspermissive for the activity of the polypeptide of claim 1, b) assessingthe activity of the polypeptide of claim 1 in the presence of the testcompound, and c) comparing the activity of the polypeptide of claim 1 inthe presence of the test compound with the activity of the polypeptideof claim 1 in the absence of the test compound, wherein a change in theactivity of the polypeptide of claim 1 in the presence of the testcompound is indicative of a compound that modulates the activity of thepolypeptide of claim
 1. 27. A method for screening a compound foreffectiveness in altering expression of a target polynucleotide, whereinsaid target polynucleotide comprises a sequence of claim 5, the methodcomprising: a) exposing a sample comprising the target polynucleotide toa compound, under conditions suitable for the expression of the targetpolynucleotide, b) detecting altered expression of the targetpolynucleotide, and c) comparing the expression of the targetpolynucleotide in the presence of varying amounts of the compound and inthe absence of the compound.
 28. A method for assessing toxicity of atest compound, said method comprising: a) treating a biological samplecontaining nucleic acids with the test compound; b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide of claim 11 underconditions whereby a specific hybridization complex is formed betweensaid probe and a target polynucleotide in the biological sample, saidtarget polynucleotide comprising a polynucleotide sequence of apolynucleotide of claim 11 or fragment thereof; c) quantifying theamount of hybridization complex; and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 29. Adiagnostic test for a condition or disease associated with theexpression of GCREC in a biological sample comprising the steps of: a)combining the biological sample with an antibody of claim 10, underconditions suitable for the antibody to bind the polypeptide and form anantibody:polypeptide complex; and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 30. The antibody of claim 10, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 31. Acomposition comprising an antibody of claim 10 and an acceptableexcipient.
 32. A method of diagnosing a condition or disease associatedwith the expression of GCREC in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 31. 33. Acomposition of claim 31, wherein the antibody is labeled.
 34. A methodof diagnosing a condition or disease associated with the expression ofGCREC in a subject, comprising administering to said subject aneffective amount of the composition of claim
 33. 35. A method ofpreparing a polyclonal antibody with the specificity of the antibody ofclaim 10 comprising: a) immunizing an animal with a polypeptide havingan amino acid sequence selected from the group consisting of SEQ IDNO:1-10, or an immunogenic fragment thereof, under conditions to elicitan antibody response; b) isolating antibodies from said animal; and c)screening the isolated antibodies with the polypeptide, therebyidentifying a polyclonal antibody which binds specifically to apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-10.
 36. An antibody produced by a method ofclaim
 35. 37. A composition comprising the antibody of claim 36 and asuitable carrier.
 38. A method of making a monoclonal antibody with thespecificity of the antibody of claim 10 comprising: a) immunizing ananimal with a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-10, or an immunogenic fragmentthereof, under conditions to elicit an antibody response; b) isolatingantibody producing cells from the animal; c) fusing the antibodyproducing cells with immortalized cells to form monoclonalantibody-producing hybridoma cells; d) culturing the hybridoma cells;and e) isolating from the culture monoclonal antibody which bindsspecifically to a polypeptide having an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1-10.
 39. A monoclonal antibodyproduced by a method of claim
 38. 40. A composition comprising theantibody of claim 39 and a suitable carrier.
 41. The antibody of claim10, wherein the antibody is produced by screening a Fab expressionlibrary.
 42. The antibody of claim 10, wherein the antibody is producedby screening a recombinant immunoglobulin library.
 43. A method fordetecting a polypeptide having an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-10 in a sample, comprising the steps of:a) incubating the antibody of claim 10 with a sample under conditions toallow specific binding of the antibody and the polypeptide; and b)detecting specific binding, wherein specific binding indicates thepresence of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-10 in the sample.
 44. A method ofpurifying a polypeptide having an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-10 from a sample, the method comprising:a) incubating the antibody of claim 10 with a sample under conditions toallow specific binding of the antibody and the polypeptide; and b)separating the antibody from the sample and obtaining the purifiedpolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-10.
 45. A polypeptide of claim 1, comprisingthe amino acid sequence of SEQ ID NO:1.
 46. A polypeptide of claim 1,comprising the amino acid sequence of SEQ ID NO:2.
 47. A polypeptide ofclaim 1, comprising the amino acid sequence of SEQ ID NO:3.
 48. Apolypeptide of claim 1, comprising the amino acid sequence of SEQ IDNO:4.
 49. A polypeptide of claim 1, comprising the amino acid sequenceof SEQ ID NO:5.
 50. A polypeptide of claim 1, comprising the amino acidsequence of SEQ ID NO:6.
 51. A polypeptide of claim 1, comprising theamino acid sequence of SEQ ID NO:7.
 52. A polypeptide of claim 1,comprising the amino acid sequence of SEQ ID NO:8.
 53. A polypeptide ofclaim 1, comprising the amino acid sequence of SEQ ID NO:9.
 54. Apolypeptide of claim 1, comprising the amino acid sequence of SEQ IDNO:10.
 55. A polynucleotide of claim 11, comprising the polynucleotidesequence of SEQ ID NO:11.
 56. A polynucleotide of claim 11, comprisingthe polynucleotide sequence of SEQ ID NO:12.
 57. A polynucleotide ofclaim 11, comprising the polynucleotide sequence of SEQ ID NO:13.
 58. Apolynucleotide of claim 11, comprising the polynucleotide sequence ofSEQ ID NO:14.
 59. A polynucleotide of claim 11, comprising thepolynucleotide sequence of SEQ ID NO:15.
 60. A polynucleotide of claim11, comprising the polynucleotide sequence of SEQ ID NO:16.
 61. Apolynucleotide of claim 11, comprising the polynucleotide sequence ofSEQ ID NO:17.
 62. A polynucleotide of claim 11, comprising thepolynucleotide sequence of SEQ ID NO:18.
 63. A polynucleotide of claim11, comprising the polynucleotide sequence of SEQ ID NO:19.
 64. Apolynucleotide of claim 11, comprising the polynucleotide sequence ofSEQ ID NO:20.